Binding moieties for biofilm remediation

ABSTRACT

Binding agents able to disrupt bacterial biofilms of diverse origin are described, including monoclonal antibodies suitable for administration to a selected species, and antibody mimics including aptamer nucleic acids. Methods to prevent formation of or to dissolve biofilms with these binding agents are also described. Immunogens for eliciting antibodies to disrupt biofilms are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 14/789,842filed 1 Jul. 2015, which is a continuation-in-part of U.S. Ser. No.14/668,767 filed 25 Mar. 2015, which is a continuation-in-part of U.S.Ser. No. 14/497,147 filed 25 Sep. 2014, which claimed priority fromprovisional application Ser. No. 61/926,828 filed 13 Jan. 2014. Thisapplication is also a continuation-in-part of U.S. Ser. No. 15/042,061filed 11 Feb. 2016. The contents of these documents are incorporatedherein by reference.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission as ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 388512013122 SeqList.txt,date recorded: 28 Apr. 2016, size: 65,499 bytes).

TECHNICAL FIELD

The invention relates to methods and compositions for preventingformation of or achieving dissolution of biofilms that inhibit immuneresponses and make bacteria resistant to antibiotics. More specifically,it concerns monoclonal antibodies that are derived from human cells orfrom transgenic animals expressing human antibody genes or that arehumanized forms of antibodies native to other species wherein theaffinity for a family of proteins that are implicated in the structuralintegrity of such biofilms exceeds the affinity of these proteins forbiofilm components. Monoclonal antibodies in general and otherhomogeneous binding moieties with this property are also included.

BACKGROUND ART

It is well understood in the art that bacterial infections may lead toformation of biofilms that protect the bacteria from the immune systemand lead them to enter a quiescent, slow growth state that makes themresistant to most antibiotics (Donlan, R. M., et al., Clin. Microbiol.Rev. (2002) 15:167-193). The result is persistent, recurrent infectionsthat are very difficult to eliminate. These biofilms include as a majorcomponent branched extracellular DNA molecules, whose key role wasestablished by showing that DNAse treatment reduced biofilms(Whitchurch, C. B., et al., Science (2002) 295:1487; Petersen, F. C., etal., J. Bacteriol. (2004) 186:6327). The higher order meshwork structureof the DNA molecules is achieved by specific proteins generallydesignated DNABII proteins, with homologs found in most bacterialspecies, including proteins designated as IHF (integration host factor)and HU (histone like protein) (Swinger, K. K., et al., Curr. Opin.Struct. Biol. (2004) 14:28-35; Goodman, S. D., et al., Mucosal Immunity(2011) 4:625-637). The substantial homology of these proteinsfacilitates the cooperative formation of biofilms, a feature thatfurther renders the bacteria problematic from a treatment perspective.Members of this class are known to be present in the extracellularenvironment (Winters, B. D., et al., Infect. Immun. (1993) 61:3259-3264;Lunsford, R. D., et al., Curr. Microbiol. (1996) 32:95-100; Kim, N., etal., J. Bacteriol. (2002) 184:6155-6162) and are known to force orstabilize bends in DNA, a key feature underlying higher order structurein other contexts (Teter, B., et al., Plasmid (2000) 43:73-84). Mutationof the ihfA gene in E. coli reduced or eliminated biofilm in vitro(Garcia-Contreras, R. (2008) PLoS ONE 3:e2394). The present invention isbased on the concept that supplying a binding moiety with sufficientlyhigh affinity for this class of proteins will extract the proteins fromthe biofilm and thereby provide an effective method of destroying thebiofilm by destroying the ability of the protein to bind and holdtogether the branched DNA. A supplied binding moiety against the DNABIIprotein may also destroy its ability to bind to other components presentin the biofilm.

The binding moieties, of which monoclonal antibodies or fragmentsthereof are an important embodiment, can be supplied directly toestablished biofilms or used to coat surfaces to provide animmuno-adsorbent for confining the DNABII protein(s) and therebysuppressing biofilm formation. Applications include treatments ofbacterial infections by systemic administration, subcutaneous, topicalor inhaled administration, as well as reduction of biofouling thataffects pipelines and other industrial equipment. Application tocorresponding biofilm associated diseases of animals is also part of thepresent invention.

PCT publication WO2011/123396 provides an extensive discussion ofbiofilms and provides for their removal by administering to a subjectpolypeptides that represent the DNABII protein itself as a whole or inpart, thus causing the organism to generate antibodies that can destroythe integrity of the biofilm. This document also provides, in thealternative, supplying the antibodies themselves, either ex vivo tobiofilms that exist outside an organism or to a subject to conferpassive protection. Antibodies to other biofilm associated proteins havesimilarly been used to interfere with biofilms (Sun, D., et al., Clin.Diagn. Lab. Immunol. (2005) 12:93-100; Shahrooei, M., et al., Infect.Immun. (2009) 77:3670-3678; Novotny, L. A., et al., Vaccine (2009)28:279-289).

The WO2011/123396 PCT application describes the use of polyclonal andmonoclonal antibodies generated against a particular DNABII protein (E.coli integration host factor (IHF)) to treat an animal model of thecommon ear infection (otitis media) and an animal model for periodontaldisease. It also describes generating active immunity by providing theprotein, or peptides representing the protein to a subject. The presentinvention provides improved agents for passive immunity. The epitopesfor two such monoclonal antibodies have been identified at the level ofindividual amino acids and are disclosed herein. One of theseantibodies, TRL1068, was disclosed by the present applicants in PCTapplication US2014/057771. The non-identical but overlapping epitopesidentify a region of the protein that is conformational with regard tothe linear sequence of the IHF/HU protein, and thereby identifiesfavorable conformational features for an immunogen intended to generatean immune response with efficacy for interfering with a biofilm.Identification of the conformational nature of the epitope is alsouseful in the design of screening reagents for discovery of monoclonalantibodies or other homogeneous agents with biofilm disrupting activityand for affinity purification of such agents.

DISCLOSURE OF THE INVENTION

The invention provides homogeneous compositions of binding moieties,such as aptamers, protein mimics of antibodies or monoclonal antibodiesor fragments thereof, that are particularly effective in binding DNABIIproteins and thus effective in dissolving biofilms. The most significantDNABII proteins are the alpha and beta subunits of IHF and the alpha andbeta subunits of HU proteins. In general, gram-positive bacteria haveeither the HU-alpha subunit or the HU-beta subunit whereas gram-negativebacteria generally have all four. However, there are exceptions—e.g., H.influenzae has three of these but not HU-beta. In the strains describedherein, P. aeruginosa has all four of these subunits whereas S. aureuscontains only HU-beta.

Thus, the invention in one aspect is directed to a binding moiety suchas a monoclonal antibody (mAb) that has affinity for at least one DNABIIprotein that exceeds the affinity of branched DNA, a component ofbiofilms, for said protein. Some affinities for non-sequence-specificDNA binding by these proteins are disclosed in Chen, C. et al, Biochem.J. (2004) 383:343-351, Aeling, K. A., et al., J. Biol. Chem. (2006)281:39236-39248 and Swinger, K. K., et al., J. mol. Biol. (2007)365:1005-1016. The affinities span a broad range, but have in common thefeature of being sufficiently weak that the DNABII protein would beexpected to desorb and re-bind to the biofilm over the course of minutesto hours. A high affinity mAb can thereby extract the protein bypreventing re-binding, leading to gradual dissolution of the biofilm.Gradual dissolution is useful for avoiding release of large numbers ofbacteria at once that could induce a deleterious cytokine stormreaction, and for avoiding release of large fragments of the biofilmthat could occlude blood vessels. One class of bindingmoieties—especially mAb—is that wherein binding is to the conformationalepitope in SEQ ID NO:80. It is particularly preferred that anyantibodies to be used systemically be compatible with mammaliansubjects, especially human subjects or feline, canine, porcine, bovine,ovine, caprine or equine subjects when proposed for use in thesesubjects. Such native mAbs or mAbs modified to more resemble theselected species—i.e., humanized or “species-ized”—have lower risk ofbeing rejected as foreign proteins, particularly upon repeatedadministration. Those actually derived from the species of interest havereduced risk of binding to other proteins in the body than mAbs fromother sources and thus pose lower toxicity risk. Also preferred is theproperty of binding with sufficient affinity so as to dissolve orprevent formation of biofilm derived from DNABII proteins originatingfrom at least two different bacterial species. In the case of mAbs, suchmAbs may be characterized as an Fv antibody, a bispecific antibody, achimeric antibody, species-ized antibody or a complete antibody, whereinsaid complete antibody comprises generic constant regions heterologousto the variable regions thereof.

In some embodiments, the mAbs comprise variable regions encoded bynucleic acid isolated from B cells of a human not immunized with DNABIIprotein or with a fragment thereof. In some embodiments, the screeningis performed by reacting antibodies secreted by said B cells withfull-length DNABII protein, especially IHF and/or HU protein.

Specific binding moieties illustrated herein contain at least the CDRregions of the heavy chains, and optionally the light chains of the mAbsTRL1068, TRL1070, TRL1087, TRL1215, TRL1216, TRL1218, TRL1230, TRL1232,TRL1242, TRL1245, TRL1330, TRL1335, TRL1337, TRL1338, TRL1341, TRL1347and TRL1361. However, other types of binding moieties, such as aptamers,modifications of antibodies such as camel type single-chain antibodiesand the like are also included within the scope of the invention.Examples of antibody mimics include scaffolds based on fibronectin,lipocalin, lens crystallin, tetranectin, ankyrin, Protein A (Ig bindingdomain). Small peptide families may also have antibody-like affinity andspecificity, including avian pancreatic peptides and conotoxins. Peptidenucleic acids, and “stapled” (cross-linked) peptides similarly providethe ability to generate high affinity binding agents with well-definedspecificity. All of these classes share the property of having a largenumber of potential binding agents, from which a single homogeneousagent is chosen. Homogeneous agents are preferred drug candidatescompared to, for example, polyclonal antisera for both ease ofmanufacturing and reduced risk of off-target binding activity that couldlead to toxicity.

The invention is further directed to a method to treat a biofilmassociated with an industrial process by using the binding moieties ofthe invention either to dissolve biofilms or prevent their formation. Inthis instance, the species origin of the mAbs is not of concern. Thesebinding moieties may also be applied topically on a subject to dissolvebiofilms characteristic of a condition in said subject or to preventtheir formation. The binding moieties may also be administeredsystemically for treatment of biofilms.

Thus, the invention further includes pharmaceutical or veterinarycompositions which comprise the binding moiety described above in anamount effective to treat or prophylactically inhibit the formation ofbiofilm due to infection in animal subjects.

In still other aspects, the invention is directed to recombinantmaterials and methods to prepare binding moieties of the invention thatare proteins, e.g., mAbs, and to improved recombinant methods to prepareDNABII proteins.

In other aspects, the invention is directed to novel expression systemsfor DNABII proteins to be used as immunogens and to methods to use theseDNABII proteins to identify an agent that reverses drug resistance inmultiple species of bacteria. In one embodiment, B cells obtained from ahuman or other mammal not immunized with DNABII protein or fragmentthereof are screened by testing the antibodies secreted by said B cellswith the full-length protein obtained. Nucleic acid isolated from saidcells may then be combined with nucleic acid encoding the relevantconstant regions to prepare the mAbs of the invention recombinantly.Alternatively, the variable regions may be prepared as Fv single-chainantibodies, for example.

The invention also relates to specific isolated peptides andpeptidomimetics that mimic the conformational epitope defined jointly bystudies using TRL1068, TRL1330 and TRL1337, as well as to methods forgenerating antibodies to DNABII proteins by using these specificpeptides or peptidomimetics as immunogens. These peptides may also beused to detect B cells that secrete desired antibodies in individualsthat have not been specifically immunized.

Synthetic compounds that mimic the epitope that bind the antibodies ofthe invention, such as TRL1068, TRL1330 and TRL1337 may also be used toscreen libraries of candidate binding moieties to identify those thatwill successfully dissolve biofilms. The binding moieties may be nucleicacids resulting in suitable aptamers or may be libraries of homologsthat can behave as binding moieties such as scaffolds based on ankyrin,tetranectin, and the like.

In still another aspect, the invention is directed to a method to treathuman or animal diseases for which biofilm causes drug resistance.Treatments include vaccination with immunogens that mimic theconformational epitope defined by the epitopes for TRL1068, TRL1330 andTRL1337, as well as treating subjects with the resulting sera (orconcentrated antigen binding polyclonal antibodies from such sera), orwith specific monoclonal antibodies developed from the B cells of suchimmunized animals. Examples of medical indications include: heart valveendocarditis (for which surgical valve replacement is required in thesubstantial fraction of cases that cannot be cured by high doseantibiotics due to the resistance associated with biofilm), chronicnon-healing wounds (including venous ulcers and diabetic foot ulcers),ear and sinus infections, urinary tract infections, pulmonary infections(including subjects with cystic fibrosis or chronic obstructivepulmonary disease), catheter associated infections (including renaldialysis subjects), subjects with implanted prostheses (including hipand knee replacements), and periodontal disease.

This method is effective in mammalian subjects in general, and thus isalso applicable to household pets, including periodontal disease in dogswhich is difficult to treat due to biofilm (Kortegaard, H. E., et al.,J. Small Anim. Pract. (2008) 49:610-616). Similarly, the invention hasutility for treating farm animals, including dairy cattle with mastitisdue to bacterial infections (Poliana de Castro Melo, et al., BrazilianJ. Microbiology (2013) 44:119-124). For the veterinary indications inparticular, hyperimmunization of donor animals provides a cost effectiveroute to large amounts of serum suitable for use in localized deliveryto other members of the same animal species, e.g., to the gums forperiodontal disease or to the udders for mastitis. Extraction of antigenbinding antibodies from serum provides a more concentrated source ofantibodies for this purpose. For such extraction, substantial quantitiesof the DNABII protein are needed, which can be provided by therecombinant production method disclosed herein. Alternatively, mimics ofthe conformational epitope can be used for such extraction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows ELISA results for various mAbs with respect to binding to aset of overlapping peptides derived from S. aureus DNABII protein,HU-beta, identifying multiple regions susceptible to antibodyrecognition.

FIG. 2 provides sequences and shows the locations of the empiricallydefined epitopes of key mAbs of the invention in DNABII (IHF/HU)proteins of various bacterial species SEQ ID NO:81-SEQ ID NO:91.

FIG. 3 shows that the key residues within the empirically definedepitope represented by SEQ ID NO:80 required for binding, as determinedby alanine substitution at each residue, reside on an anti-parallel betasheet conformationally restricted region of the protein.

FIG. 4 shows binding for various mAbs to the full length DNABII protein,HU-beta, from S. aureus.

FIGS. 5A and 5B show the results of ELISA assays to determine affinityof TRL1068 and TRL1330 for biofilm forming proteins derived fromdifferent bacterial strains.

FIG. 6A shows Staphylococcus aureus (Sa) biofilm treated for 12 hourswith a no antibody control (growth control) or with TRL1068 at 1.2 μg/mL(˜7 nM), a native human mAb against a conserved epitope on DNABIIproteins. TRL1068 caused dissolution of the biofilm, as evident at bothlow (500×) and high (2500×) magnification (scanning electron microscopeimages). FIG. 6B shows the parallel experiment on Pseudomonas aeruginosa(Pa) biofilm.

FIGS. 7A-7C show the results of ELISA assays to determine affinity ofTRL1068 as a function of pH for binding to HU-beta from Staphylococcusaureus and IHF-alpha from Pseudomonas aeruginosa and Klebsiellapneumoniae respectively. As shown, the binding activity is consistent inthe range of pH 4.5-pH 7.5 but drops off as the pH is lowered to 3.5 or2.5.

FIG. 8A shows the reduction in planktonic bacteria (released from thebiofilm) and FIG. 8B shows the reduction in adherent bacteria (stillretained in the biofilm) following post-infection treatment with TRL1068in a murine model for established infection on a plastic implant. ThemAb treatment combined with a standard antibiotic, daptomycin (DAP) iscompared to treatment with DAP alone. Marked reduction in bacterialcounts for the combination therapy is evident. TRL1068 was administeredsystemically at 15 mg/kg or by direct injection into the lumen of theimplanted plastic perforated cage. Similar intra-cage antibodyconcentration was achieved for both routes of administration.

MODES OF CARRYING OUT THE INVENTION

The invention includes various binding moieties of a monoclonal orhomogeneous nature that can dissolve biofilms. “Monoclonal” means thatthe binding moieties can form a homogeneous population analogous to thedistinction between monoclonal and polyclonal antibodies. In oneimportant embodiment, the exemplified binding moieties are mAbs orfragments thereof. In most embodiments, the binding moieties haveaffinity for at least one DNABII protein in the low nanomolarrange—i.e., the Kd is in the range of 10 nM-100 nM including theintervening values, such as 25 nM or 50 nM, but may also be <10 nM orless than 100 pM or less than 40 pM as preferred embodiments.

These affinities should be, in some embodiments, characteristic of theinteraction with the biofilm-forming proteins derived from amultiplicity of bacterial species, at least two, three, four or moreseparate species. In some embodiments, particularly high affinitiesrepresented by values less than 100 pM or less than 40 pM are exhibitedacross at least three species, and in particular wherein these speciesare Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiellapneumoniae. However, assurance of binding across multiple species canalso be achieved by exhibiting a high affinity with respect to anepitope that is highly conserved across multiple species. As describedbelow, the epitope for both TRL1068 and TRL1330 has been mapped towithin residues corresponding to SEQ ID NO:80 of Staphylococcus aureus,HU-beta which is in the most highly conserved part of the protein amongbacterial species (FIG. 2).

As noted in the examples below, the invention includes a number ofspecific antibodies that have been shown to bind with high affinity tovarious epitopes of Staphylococcus aureus HU-beta. These include, inaddition to TRL1068, mAbs designated TRL1070, TRL1087, TRL1215, TRL1216,TRL1218, TRL1230, TRL1232, TRL1245, TRL1330, TRL1335, TRL1337, TRL1338,TRL1341, TRL1347, TRL1361 and antibodies that compete with each of thesefor binding to a DNABII protein.

For use in treatment of bacterial infection in humans, the bindingmoieties of the invention should have at least three characteristics inorder to be maximally useful: the binding moiety should be compatiblewith the treated species—e.g., in the case of monoclonal antibodies fortreating humans, either human or humanized. The binding moiety shouldhave an affinity for the biofilm-forming DNABII protein that exceeds theaffinity of that protein for other components of the biofilm thatincludes this DNABII protein, and it should be crossreactive across theDNABII homologs from multiple bacterial species, minimally two or threesuch species including both Gram positive and Gram negative species, butpreferably a greater number, such as four, five or six or more.

Similar characteristics are relevant for use of the binding moieties ofthe invention for treatment of conditions in other species. In thiscase, the antibodies are compatible with the species in question. Thus,the antibodies may be derived from feline, canine, equine, bovine,caprine, ovine or porcine species or may be adapted from antibodies ofother animals. Analogous to “humanized,” these antibodies could becalled “species-ized” so that the relevant species is adequatelyaddressed. Alternatively, a functionally restricted polyclonal antibodypreparation can be prepared by generation of antibodies in serum ofsubjects immunized with peptides that mimic the conformational epitopedefined by human mAbs TRL1068, TRL1330 and TRL1337, with furthernarrowing of the specificity accomplished by affinity purification usingeither intact DNABII protein or an epitope mimic. This approach is moreappropriate for veterinary indications than for human use.Hyperimmunization of animals to generate the serum is ethicallyacceptable, but is not feasible for humans; and the risk of adverseevents from a polyclonal antiserum is more acceptable for treatment ofanimals than for treatment of humans. Indeed, as exemplified below,suitable B cells can be screened for antibodies immunoreactive withfull-length DNABII proteins in humans that have not been immunized witheither the DNABII protein itself or with a fragment thereof. In additionto antisera per se, specific monoclonal antibodies can be prepared byscreening B cells of the subject for secretion of antibodies that areimmunoreactive with, for example, the full-length protein in questionwhich contains the epitope from which the mimic was derived. Concurrentscreening with more than one DNABII protein can assist in identifyingmAbs that show cross-species binding. Nucleic acid contained insuccessful B cells can then be extracted and used to prepare monoclonalantibodies recombinantly.

As the illustrative antibodies disclosed herein in the examples belowcontain variable regions that are derived from humans, and constantregions which are cloned independently of said variable regions but arealso derived from humans, these antibodies offer particular advantagesfor repeated use in humans. When the subject to be administered the mAbis non-human, it is advantageous for repeated use to administer nativemAbs similarly derived from that species. Alternatively, an equivalentof the human variable regions, optionally fused to an Fc region from thehost species to be treated, may be used. This variable region may be, insome embodiments, an Fab portion or a single-chain antibody containingCDR regions from both the heavy and light chains or heavy chain only.Bispecific forms of these variable regions equivalents can also beconstructed, with numerous constructs described in the literature.Although the typical “mAb” will be a protein or polypeptide (“proteins,”“polypeptide” and “peptide” are used interchangeably herein withoutregard to length) for use in subjects, the mAbs may also be supplied viadelivery of nucleic acids that then generate the proteins by in situtranslation in cells of the subject. In addition, nucleic acid moleculesthat mimic the binding characteristics of these polypeptides or proteinscan be constructed—i.e., aptamers can be constructed to bind moleculesthat are identified as described below by their ability to mimic thebinding moieties. Successful mimicry of these aptamers for theprotein-based binding moieties can be verified both biochemically andfunctionally to confirm that the affinity of the aptamer is sufficientfor therapeutic efficacy. In light of the well-defined conformationallyconstrained region that includes the epitopes for TRL1068, TRL1330 andTRL1337, preparation of such aptamers is straightforward using wellestablished methods (Lin, H., et al., Biomicrofluidics (2014) 8:041501).

In more detail, in one embodiment, the aptamers or other bindingmoieties such as alternative antibody scaffolds can be identified byscreening libraries comprising candidate aptamers or alternative bindingmoieties with mimics of epitopes that bind, specifically, the mAbs ofthe invention shown to be successful herein—e.g., TRL1068 or TRL1330. Inone embodiment, for example, TRL1068 can itself be used to screenlibraries of candidate mimics and compounds that successfully bindTRL1068 can then, in turn, be used to screen libraries of candidateaptamers or alternative binding moieties.

With respect to protein-based monoclonal binding moieties, in additionto typical monoclonal antibodies or fragments thereof that areimmunologically specific for the same antigen, various forms of otherscaffolding, including single-chain antibody forms such as those derivedfrom camel, llama or shark could be used as well as antibody mimicsbased on other scaffolds such as fibronectin, lipocalin, lenscrystallin, tetranectin, ankyrin, Protein A (Ig binding domain), or thelike. Short structured peptides may also be used if they providesufficient affinity and specificity, e.g., peptides based on inherentlystable structures such as conotoxins or avian pancreatic peptides, orpeptidomimetics that achieve stable structures by crosslinking and/oruse of non-natural amino acids (Josephson, K., et al., J. Am. Chem. Soc.(2005) 127:11727-11725). In general, “monoclonal antibody (mAb)”includes all of the foregoing. As for aptamers, generation of suchmolecules is straightforward using well established methods.

As used herein, the term “antibody” includes immunoreactive fragments oftraditional antibodies even if, on occasion, “fragments” are mentionedredundantly. The antibodies, thus, include Fab, F(ab′)₂, F_(v)fragments, single-chain antibodies which contain substantially onlyvariable regions, bispecific antibodies and their various fragmentedforms that still retain immunospecificity and proteins in general thatmimic the activity of “natural” antibodies by comprising amino acidsequences or modified amino acid sequences (i.e., pseudopeptides) thatapproximate the activity of variable regions of more traditionalnaturally occurring antibodies.

In particular, in the case of embodiments which are monoclonalantibodies, fully human antibodies which are, however, distinct fromthose actually found in nature, are typically prepared recombinantly byconstructing nucleic acids that encode a generic form of the constantregion of heavy and/or light chain and further encode heterologousvariable regions that are representative of human antibodies. Otherforms of such modified mAbs include single-chain antibodies such thatthe variable regions of heavy and light chain are directly bound withoutsome or all of the constant regions. Also included are bispecificantibodies which contain a heavy and light chain pair derived from oneantibody source and a heavy and light chain pair derived from adifferent antibody source. Similarly, since light chains are ofteninterchangeable without destroying specificity, antibodies composed of aheavy chain variable region that determines the specificity of theantibody combined with a heterologous light chain variable region areincluded within the scope of the invention. Chimeric antibodies withconstant and variable regions derived, for example, from differentspecies are also included.

For the variable regions of mAbs, as is well known, the critical aminoacid sequences are the CDR sequences arranged on a framework whichframework can vary without necessarily affecting specificity ordecreasing affinity to an unacceptable level. Definition of these CDRregions is accomplished by art-known methods. Specifically, the mostcommonly used method for identifying the relevant CDR regions is that ofKabat as disclosed in Wu, T. T., et al., J. Exp. Med. (1970) 132:211-250and in the book Kabat, E. A., et al. (1983) Sequence of Proteins ofImmunological Interest, Bethesda National Institute of Health, 323pages. Another similar and commonly employed method is that of Chothia,published in Chothia, C., et al., J. Mol. Biol. (1987) 196:901-917 andin Chothia, C., et al., Nature (1989) 342:877-883. An additionalmodification has been suggested by Abhinandan, K. R., et al., Mol.Immunol. (2008) 45:3832-3839. The present invention includes the CDRregions as defined by any of these systems or other recognized systemsknown in the art.

The specificities of the binding of the mAbs of the invention aredefined, as noted, by the CDR regions mostly those of the heavy chain,but complemented by those of the light chain as well (the light chainsbeing somewhat interchangeable). Therefore, the mAbs of the inventionmay contain the three CDR regions of a heavy chain and optionally thethree CDR's of a light chain that matches it. The invention alsoincludes binding agents that bind to the same epitopes as those thatactually contain these CDR regions. Thus, for example, also included areaptamers that have the same binding specificity—i.e., bind to the sameepitopes as do the mAbs that actually contain the CDR regions. Becausebinding affinity is also determined by the manner in which the CDR's arearranged on a framework, the mAbs of the invention may contain completevariable regions of the heavy chain containing the three relevant CDR'sas well as, optionally, the complete light chain variable regioncomprising the three CDR's associated with the light chain complementingthe heavy chain in question. This is true with respect to the mAbs thatare immunospecific for a single epitope as well as for bispecificantibodies or binding moieties that are able to bind two separateepitopes, for example, divergent DNABII proteins from two bacterialspecies.

The mAbs of the invention may be produced recombinantly using knowntechniques. Thus, with regard to the novel antibodies described herein,the invention also relates to nucleic acid molecules comprisingnucleotide sequence encoding them, as well as vectors or expressionsystems that comprise these nucleotide sequences, cells containingexpression systems or vectors for expression of these nucleotidesequences and methods to produce the binding moieties by culturing thesecells and recovering the binding moieties produced. Any type of celltypically used in recombinant methods can be employed includingprokaryotes, yeast, mammalian cells, insect cells and plant cells. Alsoincluded are human cells (e.g., muscle cells or lymphocytes) transformedwith a recombinant molecule that encodes the novel antibodies.

Typically, expression systems for the proteinaceous binding moieties ofthe invention include a nucleic acid encoding said protein coupled tocontrol sequences for expression. In many embodiments, the controlsequences are heterologous to the nucleic acid encoding the protein.

Bispecific binding moieties may be formed by covalently linking twodifferent binding moieties with different specificities. For example,the CDR regions of the heavy and optionally light chain derived from onemonospecific mAb may be coupled through any suitable linking means topeptides comprising the CDR regions of the heavy chain sequence andoptionally light chain of a second mAb. If the linkage is through anamino acid sequence, the bispecific binding moieties can be producedrecombinantly and the nucleic acid encoding the entire bispecific entityexpressed recombinantly. As was the case for the binding moieties with asingle specificity, the invention also includes the possibility ofbinding moieties that bind to one or both of the same epitopes as thebispecific antibody or binding entity/binding moiety that actuallycontains the CDR regions.

The invention further includes bispecific constructs which comprise thecomplete heavy and light chain sequences or the complete heavy chainsequence and at least the CDR's of the light chains or the CDR's of theheavy chains and the complete sequence of the light chains.

The invention is also directed to nucleic acids encoding the bispecificmoieties and to recombinant methods for their production, as describedabove.

Multiple technologies now exist for making a single antibody-likemolecule that incorporates antigen specificity domains from two separateantibodies (bi-specific antibody). Thus, a single antibody with verybroad strain reactivity can be constructed using the Fab domains ofindividual antibodies with broad reactivity to diverse homologs.Suitable technologies have been described by MacroGenics (Rockville,Md.), Micromet (Bethesda, Md.) and Merrimac (Cambridge, Mass.). (See,e.g., Orcutt, K. D., et al., Protein Eng. Des. Sel. (2010) 23:221-228;Fitzgerald, J., et al., MAbs. (2011) 1:3; Baeuerle, P. A., et al.,Cancer Res. (2009) 69:4941-4944.)

The invention is also directed to pharmaceutical and veterinarycompositions which comprise as active ingredients the binding moietiesof the invention. The compositions contain suitable physiologicallycompatible excipients such as buffers and other simple excipients. Thecompositions may include additional active ingredients as well, inparticular antibiotics. It is often useful to combine the binding moietyof the invention with an antibiotic appropriate to a condition to beaddressed since the efficacy of most antibiotics is greater against theplanktonic state of the bacteria than against the sessile, biofilmembedded state. Additional active ingredients may also includeimmunostimulants and/or antipyrogenics and analgesics.

The binding moieties of the invention may also be used in diagnosis byadministering them to a subject and observing any complexation with anybiofilm present in the subject. In this embodiment the binding moietiesare typically labeled with an observable label, such as a fluorescent orchemiluminescent compound in a manner analogous to labeling withbacteria that produce luciferase for non-invasive detection as describedin Chang, H. M., et al., J. Vis. Exp. (2011) 10.3791/2547. The assay mayalso be performed on tissues obtained from the subject. The presence ofa biofilm is detected in this manner if it is present, and the progressof treatment may also be monitored by measuring the complexation overtime. The identity of the infectious agent may also be established byemploying binding moieties that are specific for a particular strain orspecies of infectious agent. For diagnostic purposes, it is particularlyfavorable to target epitopes that are not sterically occluded when theprotein is complexed with DNA. For example the epitope of TRL1361 hasbeen determined to lie outside the contact sites with DNA. Antibodiessuch as this also provide the opportunity to construct sandwichimmunoassays, wherein one antibody is used to capture the antigen and asecond antibody that binds at a different site is used to detect thecaptured antigen. Such assays provide high specificity and are common inthe field of diagnostics, with particular utility in a multiplexed assayto reduce the effect of antibody cross-reactivity to other antigens.

The invention also includes a method for identifying suitable immunogensfor use to generate antibodies which method includes assessing thebinding of the binding moieties of the invention, such as mAbs describedabove, to a candidate peptide or other epitope mimicking molecule. Thisis an effective method, not only to identify suitable immunogens, butalso to identify compounds that can be used as a basis for designingaptamers that mimic the binding moieties of the invention. The method isgrounded in the fact that if a vaccine immunogen cannot bind to anoptimally effective mAb, it is unlikely to be able to induce suchantibodies. Conversely, an immunogen that is a faithful inverse of theoptimal mAb provides a useful template for constructing a mimic of theoptimal mAb. In its simplest form, this method employs a binding moietysuch as one of the mAbs of the invention as an assay component and teststhe ability of the binding moiety to bind to a candidate immunogen in alibrary of said candidates. The invention further includesidentification of the conformationally restricted region defined by theoverlap of the epitopes for TRL1068, TRL1330 and TRL1337 as aparticularly favorable starting point for such immunogen optimization.

Thus, the binding moieties of the invention may be used in highthroughput assays to identify from combinatorial libraries of compoundsor peptides or other substances those substances that bind with highaffinity to the binding moieties of the invention. General techniquesfor screening combinatorial or other libraries are well known: Glokler,J., et al., Molecules (2010) 15:2478-2490. It may be advantageous toestablish affinity criteria by which effective candidate immunogens orother binding partners of the binding moieties of the invention can beselected. The binding moiety, then, can become a template for the designof an aptamer that will bind an epitope of the DNABII protein,preferably across a number of species, but which contains too fewnucleotides to act as a structural component in a biofilm. Thus, theresulting aptamers are composed of only 25 or less oligonucleotides,preferably 10-20 nucleotides which are sufficient to effect binding, butnot sufficient to behave as structural components for biofilms. Acorresponding number of individual monomers would be characteristic ofnucleic acid mimics, such as peptide nucleic acids as well.

In one particular example, the immunogen discussed above could be apeptide that represents an epitope to which the binding moiety istightly bound. The binding moiety may be an mAb, and this isparticularly favorable if the binding moiety or mAb is crossreactivewith regard to the DNABII protein across a number of species. Theepitope then represents a template which can form the basis for formingaptamers—i.e., short species of DNA or suitable DNA analogs such aspeptide nucleic acids which can then behave as agents to bind the DNABIIproteins thus preventing these proteins from forming the biofilms thatwould result from interaction with longer forms of DNA. Such chemicallysturdy mimics could be used, for example, to coat pipes in industrialsettings thus permitting sequestering of DNABII proteins to preventbiofilm formation. Due to the lower immunogenicity, mAbs are generallypreferable as pharmaceuticals, but such aptamer mimics are alsopotentially useful as pharmaceuticals, again, by virtue of their abilityto prevent binding of DNABII proteins to longer forms of DNA forformation of biofilms.

In addition, the ability of the binding moieties of the invention toovercome drug resistance in a variety of bacteria can be assessed bytesting the binding moieties of the invention against a panel or libraryof DNABII proteins from a multiplicity of microbial species. Bindingmoieties that are able to bind effectively a multiplicity of suchproteins are thus identified as suitable not only for dissolvingbiofilms in general, but also as effective against a variety ofmicrobial strains. It is also useful to identify binding moieties thathave utility in acidic environments wherein the affinity of a candidatebinding moiety for a DNABII protein over a range of pH conditions istested and moieties with a low nanomolar affinity at pH 4.5 areidentified as having utility in acidic environments.

The binding moieties of the invention are also verified to have anaffinity with respect to at least one DNABII protein greater than theaffinity of a biofilm component for the DNABII protein which comprisescomparing the affinity of the binding moiety for the DNABII proteinversus the affinity of a component of the biofilm, typically branchedDNA, for the DNABII protein. This can be done in a competitive assay, orthe affinities can be determined independently.

The DNABII proteins used in these assays may be prepared in mammaliancells at relatively high yield, thereby overcoming difficulties inexpressing these proteins in bacteria. Specifically, the full-lengthDNABII proteins of the invention can be prepared for any purpose,including as reagents for screening B cells for antibody secretion byculturing mammalian cells comprising suitable expression vectors thatinclude nucleic acids encoding the full-length forms of these proteins.One aspect of the invention is the successful production of full-lengthDNABII proteins by recombinant production in mammalian cell culture.

All of the assays above involve assessing binding of two prospectivebinding partners in a variety of formats.

A multitude of assay types are available for assessing successfulbinding of two prospective binding partners. For example, one of thebinding partners can be bound to a solid support and the other labeledwith a radioactive substance, fluorescent substance or a colorimetricsubstance and the binding of the label to the solid support is testedafter removing unbound label. The assay can, of course, work either waywith the binding moiety attached to the solid support and a candidateimmunogen or DNABII protein labeled or vice versa where the candidate isbound to solid support and the binding moiety is labeled. Alternatively,a complex could be detected by chromatographic or electrophoretic meansbased on molecular weight such as SDS-page. The detectable label in thecontext of the binding assay can be added at any point. Thus, if, forexample, the mAb or other binding moiety is attached to a solid supportthe candidate immunogen can be added and tested for binding by supplyinga labeled component that is specific for the candidate immunogen.Hundreds of assay formats for detecting binding are known in the art,including, in the case where both components are proteins, the yeasttwo-hybrid assay.

In addition to this straightforward application of the utility of thebinding moieties of the invention, the identification of a suitablepowerful immunogen can be determined in a more sophisticated series ofexperiments wherein a panel of mAbs against the DNABII protein isobtained and ranked in order by efficacy. A full suite of antibodies orother binding moieties can be prepared against all possible epitopes byassessing whether additional binding moieties compete for binding withthe previous panel of members. The epitopes for representative bindingmAbs for each member of the complete suite can be accomplished bybinding to a peptide array representing the possible overlappingepitopes of the immunogen or by X-ray crystallography, NMR orcryo-electron microscopy. An optimal vaccine antigen would retain thespatial and chemical properties of the optimal epitope defined as thatrecognized by the most efficacious mAbs as compared to less efficaciousmAbs but does not necessarily need to be a linear peptide. It maycontain non-natural amino acids or other crosslinking motifs.

Thus, even beyond the specific mAbs set forth herein, optimal immunogenscan be obtained, which not only are useful in active vaccines, but alsoas targets for selecting aptamers. Specifically, in addition to thepeptide of SEQ ID NO:80, positions 5-20 of peptides of SEQ ID NOS:71-73and 75-79 are identified.

Another aspect of the invention is a method to prepare higher yields ofthe bacterial/microbial DNABII proteins which are typically difficult toexpress at high levels in bacteria possibly due to toxicity to thosecells. The standard method for preparation of these proteins isdescribed by Nash, H. A., et al., J. Bacteriol (1987) 169:4124-4127 whoshowed that the IHF of E. coli could be effectively prepared if bothchains of said protein (IHF alpha and IHF beta) are produced in the sametransformant. Applicants have found that they are able to obtain higheryields, as much as 5-10 mg/l of IHF, by producing homodimers transientlyin the mammalian cell line HEK293 using a widely available expressionvector. Similar results are obtained for DNABII proteins in general. Theexpression of bacterial proteins that are toxic at high levels inbacteria is conveniently achieved in mammalian cells especially forthose without glycosylation sites that would result in modification ofthe proteins when thus expressed. If tagged with a polyhistidine,purification of the resulting protein can be readily achieved.

Applications

The binding moieties of the invention including antibodies are useful intherapy and prophylaxis for any subject that is susceptible to infectionthat results in a biofilm. Thus, various mammals, such as bovine, ovineand other mammalian subjects including horses and household pets andhumans will benefit from the prophylactic and therapeutic use of thesemAbs.

The binding moieties of the invention may be administered in a varietyof ways. The peptides based on CDR regions of antibodies, includingbispecific and single chain types or alternate scaffold types, may beadministered directly as veterinary or pharmaceutical compositions withtypical excipients. Compositions that comprise micelles or othernanoparticles of various types may be useful for extending the durationof treatment. Aptamers that behave as binding agents similar to mAbs canbe administered in the same manner. Further, the binding agent may beconjugated to any of the solid supports known in the literature, such asPEG, agarose or a dextran, to function as an immuno-sorbent forextracting DNABII from a biofilm. Alternatively, the peptide-based mAbsmay be administered as the encoding nucleic acids either as naked RNA orDNA or as vector or as expression constructs. The vectors may benon-replicating viral vectors such as adeno associated virus vectors(AAV) or the encoding nucleic acid sequence may be chemically orphysically delivered into cells (Suskovitch, T. J., and Alter, G.,Expert Rev Vaccines (2015) 14:205-219). Use of nucleic acids as drugs asopposed to their protein counterparts is helpful in controllingproduction costs.

These are administered in a variety of protocols, including intravenous,subcutaneous, intramuscular, topical (particularly for chronicnon-healing wounds and periodontal disease), inhaled and oral or bysuppository. Similar routes of administration can be used with regard tothe binding moieties themselves. One useful way to administer thenucleic acid-based forms of either the binding moieties themselves(aptamers) or those encoding the protein form of binding moieties isthrough a needleless approach such as the agro-jet needle-free injectordescribed in US2001/0171260.

The peptides that include the epitopes of the high affinity,cross-species binding human mAbs against DNABII proteins as describedherein are also useful as active components of vaccines to stimulateimmunogenic responses which will generate antibodies in situ fordisruption of biofilms. The types of administration of these immunogensor peptidyl mimetics that are similarly effective are similar to thosefor the administration of binding moieties, including various types ofantibodies, etc. Liposomal compositions that comprise micelles or othernanoparticles of various types may also be used to enhanceimmunogenicity. The peptidomimetics may themselves be in the form ofaptamers or alternative structures that mimic the immunogenic peptidesdescribed herein. For those immunogens, however, that are proteins orpeptides, the administration may be in the form of encoding nucleicacids in such form as will produce these proteins in situ. Theformulation, routes of administration, and dosages are determinedconventionally by the skilled artisan.

As shown in the examples below, two antibodies of the invention,TRL1068, TRL1330 and TRL1337 have particularly favorable affinity andspecificity characteristics for the DNABII protein. Therefore,immunogens that bind these specific antibodies will be particularlypowerful in generating effective antisera. By analyzing overlappingpeptides and alanine replacements, it has been found that the epitopesfor both of these highly binding antibodies are conformational epitopeslocated on a short anti-parallel beta sheet connected by a loop (a “betahairpin”). These are shown in FIG. 4. Since these structures are nowknown, it is well within the skill of the art to construct peptidylmimetics which preserve the conformation of the sequence, for example bycrosslinking to lock in the conformation, and render it more effectivethan simply the linear peptide representing the epitope itself and tosubstitute, for example, non-natural amino acids or other than peptidebonds to render the immunogen more stable and resistant to hydrolysis.It is also possible to design alternative non-peptide mimics includingpeptide nucleic acids that have essentially the same shape and bindingcharacteristics as the now identified immunogen. Focusing on the shortstretch of beta sheet defined herein is advantagous for constructingepitope mimics.

As set forth in Example 2 below, as the epitopes to which TRL1068 andTRL1330 bind are in the form of a conformational epitope, methods areavailable in the art to provide peptidomimetics that are useful asimmunogens. Such methods are disclosed in (Jimenez, M. A., Methods Mol.Biol. (2014) 1216:15-52), including design of artificial crosslinkers tostabilize the structure (Celentano, V., et al., Chem. Commun (Camb.)(2012) 48:762-764). Mimetics of this type of structure have beengenerated that mimic the binding of HIV Tat protein to RNA with low nMaffinity (Athanassiou, Z., et al., Biochemistry (2007) 46:741-751), anda beta hairpin mimic has been described that binds to VEGF receptor-2with low nM affinity (Patel, S., et al., Protein Eng. Des. Sel. (2013)26:307-315). Construction of mimics is also described in Schmidt, J., etal., Bioorganic. Med. Chem. (2013) 21:5806-5810 and in Lesniak, W. G.,et al., Mol. Pharm. (2015) 12:941-953. Briefly, small libraries ofcrosslinked or cyclic compounds based on the structure of the betahairpin can be constructed and evaluated for structure/activityrelationships by means of competition assays wherein the stabilized betahairpin structure that includes the epitopes is used to compete withcandidate peptidomimetics binding to the relevant antibody, e.g.,TRL1068. Those candidates that successfully compete with the nativeepitope are selected as suitable immunogens.

The types of conditions for which the administration either of thevaccine type for active generation of antibodies for biofilm control orfor passive treatment by administering the antibodies, per se, includeany condition that is characterized by or associated with the formationof biofilms. These conditions include: heart valve endocarditis, bothnative and implanted (for which a substantial fraction of cases cannotbe cured by high dose antibiotics due to the resistance associated withbiofilm), chronic non-healing wounds (including venous ulcers anddiabetic foot ulcers), ear and sinus infections, urinary tractinfections, pulmonary infections (including subjects with cysticfibrosis or chronic obstructive pulmonary disease), catheter associatedinfections (including renal dialysis subjects), subjects with implantedprostheses (including hip and knee replacements), and periodontaldisease.

One particular condition for which biofilms have been implicated is Lymedisease. It has been shown that the relevant bacteria can form a biofilmin vitro and this is thought to be a substantive contributor to theprolonged course of the disease and resistance to antibiotics. Theincidence is more than 30,000 cases per year in the U.S. An alignment ofthe HU (single gene) from Borrelia burgdorferi which is the causativebacteria shows high similarity to other IHF/HU genes in the putativeepitope. Thus, the treatment of Lyme disease specifically as anindication is a part of the invention. The isolation of B. burgdorferigenes encoding HU was described by Tilly, K., et al., Microbiol. (1996)142:2471-2479 and characterization of the biofilm formed by theseorganisms in vitro was described by Sapi, E., et al., PLoS 1 (2013)7:e1848277. Similar biofilms have been observed in vivo by Sapi, E., etal., Eur. J. Microbiol. and Immunol. (2016) 6(1):9-24.

For use in diagnosis, the binding moieties can be used to detectbiofilms in vivo by administering them to a subject or in vitro usingtissue obtained from the subject. Detection of complexation demonstratesthe presence of biofilm. Detection is facilitated by conjugating thebinding moiety to a label, such as a fluorescent, chemiluminescent orradioactive label, or in the case of in vitro testing, with an enzymelabel. Many such fluorescent, chemiluminescent, radioactive and enzymelabels are well known in the art. Treatment course can also be monitoredby measuring the disappearance of biofilm over time. The diagnosticapproach enabled by the invention is much less complex than currentmethods for, for example, endocarditis, where the current diagnostic istrans-esophageal echocardiogram. In addition, the detection/quantitationmethod can be used in evaluating the effectiveness of compounds indissolving or inhibiting the formation of biofilms in laboratorysettings. Conjugates of the binding moieties of the invention withdetectable labels are generally useful in detection and/or quantitationof biofilms in a variety of contexts.

A particularly useful antibody for imaging is an antibody that binds toan epitope on the DNABII protein that does not interact with DNA sincethe binding will not require competition with DNA for association withthe biofilm. As shown in Example 2 below, TRL1361 is such an antibodyand is particularly useful in the detection methods set forth in theprevious paragraph.

In addition, since it is now clear that antibodies are available thatbind different epitope sites, sandwich assays become available wherein,for example, TRL1068 or TRL1330 or an antibody that binds an epitopeother than that bound by TRL1361 is used as either the capture orlabeling antibody and TRL1361 is used as the opposite member—forexample, wherein TRL1361 is used as a capture antibody and TRL1068 isprovided with a label.

As noted above, the binding moieties of the invention are not limited intheir utility to therapeutic (or diagnostic) uses, but can be employedin any context where a biofilm is a problem, such as pipelines or otherindustrial settings. The mode of application of these binding moietiesto the biofilms in these situations, again, is conventional.

For example, surfaces associated with an industrial or other setting canbe coated with the binding moieties of the invention including theaptamers described above. This effects absorption of the DNABII proteinand prevents formation of biofilms. The binding moieties of theinvention may also be applied to the biofilms directly to effectdissolution.

The following examples are offered to illustrate but not to limit theinvention.

EXAMPLE 1 Preparation of Antibodies

Human peripheral blood mononuclear cells (PBMC's) were obtained fromanonymized blood bank donors, under informed consent. The cells weresubjected to the CellSpot™ assay, a micro-assay that detects cells thatsecrete antibodies that bind a defined antigen, to determine theirability to bind the DNABII protein derived from one or more bacterialspecies. The DNABII protein used to screen was full length DNABIIprepared in mammalian cells. The full length proteins used were HU fromStaphylococcus aureus; IHF from Klebsiella pneuomoniae; and IHF fromHaemophilus influenza. The CellSpot™ assay is described in U.S. Pat.Nos. 7,413,868 and 7,939,344. After isolating the B cells from wholeblood, they were stimulated with cytokines and mitogens to initiate abrief period of proliferation and antibody secretion (lasting ˜5 days)and plated for subjection to the assays; the encoding nucleic acids forthe variable regions were extracted and used to produce the antibodiesrecombinantly following fusion of the variable region encoding DNA toDNA cloned independently that codes for the constant region of theantibody.

Antibodies were selected from over 5 million B cells based on binding toat least one of the DNABII proteins screened. These antibodies: TRL1068,TRL1070, TRL1087, TRL1215, TRL1216, TRL1218, TRL1230, TRL1232, TRL1242,TRL1245, TRL1330, TRL1335, TRL1337, TRL1338, TRL1341, TRL1347 andTRL1361 were characterized by affinity as measured by adsorption ELISA.

This result establishes the feasibility of a focused screen to isolatehigh affinity, cross-strain binding antibodies. Recovery of suchantibodies from human blood is unexpected, since no immunogen wasprovided, and the low frequency of such mAbs underscores theunpredictability of their presence. As described in PCT WO2011/123396(FIG. 13), IHF complexed to DNA induces a robust immune response that isdirected towards specific regions of IHF that are not protective whereasthe immune response to IHF not complexed to DNA induced the formation ofhighly protective antibodies. The natural state of the protein is in theform of a complex with DNA; that is, the protein is a limiting factorfor biofilm formation since addition of exogenous protein increases theamount of biofilm formed (Devaraj, A., et al., Mol. Microbiol. (2015 Mar11) doi: 10.1111/mmi.12994. Epub ahead of print). Thus, as described inPCT WO2011/123396 the sites that induce a protective response arenormally masked by DNA. Despite these facts, several of the mAbsdescribed here that were cloned from human blood bind to sites that arepresumed to be in contact with DNA. No immunization with isolated IHF orpeptides thereof was required to stimulate the production of suchantibodies.

-   -   TRL1068 heavy chain variable region has the amino acid sequence:

(SEQ ID NO: 1) QVQLVESGPGLVKPSETLSLTCRVSGDSNRPSYWSWIRQAPGKAMEWIGYVYDSGVTIYNPSLKGRVTISLDTSKTRFSLKLTSVIAADTAVYYCARERF DRTSYKSWWGQGTQVTVSS;

-   -   TRL1068 light chain variable region has the amino acid sequence:

(SEQ ID NO: 2) DIVLTQAPGTLSLSPGDRATLSCRASQRLGGTSLAWYQHRSGQAPRLILYGTSNRATDTPDRFSGSGSGTDFVLTISSLEPEDFAVYYCQQYGSPPYTFG QGTTLDIK;

-   -   TRL1070 heavy chain variable region has the amino acid sequence:

(SEQ ID NO: 3) QVQLVQSGGTLVQPGGSLRLSCAASGFTFSYYSMSWVRQAPGKGLEWVANIKHDGTERNYVDSVKGRFTISRDNSEKSLYLQMNSLRAEDTAVYYCAKYYYGAGTNYPLKYWGQGTRVTVSS;

-   -   TRL1070 light chain kappa variable region has the amino acid        sequence:

(SEQ ID NO: 4) DILMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPLTFGG GTKVEIKR;

-   -   TRL1087 heavy chain variable region has the amino acid sequence:

(SEQ ID NO: 5) QVQLLESGPGLVRPSDTLSLTCTFSADLSTNAYWTWIRQPPGKGLEWIGYMSHSGGRDYNPSFNRRVTISVDTSKNQVFLRLTSVTSADTAVYFCVREVG SYYDYWGQGILVTVSS;

-   -   TRL1087 light chain kappa variable region has the amino acid        sequence:

(SEQ ID NO: 6) DIEMTQSPSSLSASVGDRITITCRASQGISTWLAWYQQKPGKAPKSLIFSTSSLHSGVPSKFSGSGSGTDFTLTITNLQPEDFATYYCQQKWETPYSFGQ GTKLDMIR;

-   -   TRL1215 heavy chain variable region has the amino acid sequence:

(SEQ ID NO: 7) QVQLVESGTEVKNPGASVKVSCTASGYKFDEYGVSWVRQSPGQGLEWMGWISVYNGKTNYSQNFQGRLTLTTETSTDTAYMELTSLRPDDTAVYYCATDK NWFDPWGPGTLVTVSS;

-   -   TRL1215 light chain lambda variable region has the amino acid        sequence:

(SEQ ID NO: 8) DIVMTQSPSASGSPGQSITISCTGTNTDYNYVSWYQHHPGKAPKVIIYDVKKRPSGVPSRFSGSRSGNTATLTVSGLQTEDEADYYCVSYADNNHYVFGS GTKVTVL;

-   -   TRL1216 heavy chain variable region has the amino acid sequence:

(SEQ ID NO: 9) QVQLVESGGGVVQPGGSLRVSCAASAFSFRDYGIHWVRQAPGKGLQWVAVISHDGGKKFYADSVRGRFTISRDNSENTLYLQMNSLRSDDTAVYYCARLVASCSGSTCTTQPAAFDIWGPGTLVTVSS;

-   -   TRL1216 light chain lambda variable region has the amino acid        sequence:

(SEQ ID NO: 10) DIMLTQPPSVSVSPGQTARITCSGDALPKKYTYWYQQKSGQAPVLLIYEDRKRPSEIPERFSAFTSWTTATLTITGAQVRDEADYYCYSTDISGDIGVFG GGTKLTVL;

-   -   TRL1218 heavy chain variable region has the amino acid sequence:

(SEQ ID NO: 11) QVQLLESGADMVQPGRSLRLSCAASGFNFRTYAMHWVRQAPGKGLEWVAVMSHDGYTKYYSDSVRGQFTISRDNSKNTLYLQMNNLRPDDTAIYYCARGL TGLSVGFDYWGQGTLVTVSS;

-   -   TRL1218 light chain lambda variable region has the amino acid        sequence:

(SEQ ID NO: 12) DIVLTQSASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVTTRPSGVSDRFSGSKSGNTASLTISGLQAEDEADYYCSSYSSGSTPA LFGGGTQLTVL;

-   -   TRL1230 heavy chain variable region has the amino acid sequence:

(SEQ ID NO: 13) QVQLVQSGGGLVKPGGSLRLSCGASGFNLSSYSMNWVRQAPGKGLEWVSSISSRSSYIYYADSVQGRFTISRDNAKNSLYLQMNSLRAEDTAIYYCARVSPSTYYYYGMDVWGQGTTVTVSS;

-   -   TRL1230 light chain lambda variable region has the amino acid        sequence:

(SEQ ID NO: 14) DIVLTQPSSVSVSPGQTARITCSGDELPKQYAYWYQQKPGQAPVLVIYKDNERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCQSADSSGTYVVFG GGTKLTVL;

-   -   TRL1232 heavy chain variable region has the amino acid sequence:

(SEQ ID NO: 15) QVQLVESGAEVKKPGALVKVSCKASGYTFSGYYMHWVRQAPGQGLEWMGWINPKSGGTKYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYFCARGGPSNLERFLERLQPRYSYDDKYAMDVWGQGTTVTVSS;

-   -   TRL1232 light chain kappa variable region has the amino acid        sequence:

(SEQ ID NO: 16) DIVMTQSPGTLSLSPGARATLSCRASQSVSSIYLAWYQQKPGQAPRLLIFGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPYTFG QGTKLEIKR;

-   -   TRL1242 heavy chain variable region has the amino acid sequence:

(SEQ ID NO: 17) QVQLVQSGTEVKKPGESLKISCEGSRYNFARYWIGWVRQMPGKGLDWMGIIYPGDSDTRYSPSFQGQVSISADKSISTAYLQWNSLKASDTAMYYCARLGSELGVVSDYYFDSWGQGTLVTVSS;

-   -   TRL1242 light chain kappa variable region has the amino acid        sequence:

(SEQ ID NO: 18) DIVLTQSPDSLAVSLGERATINCKSSQSVLDRSNNKNCVAWYQQKPGQPPKLLIYRAATRESGVPDRFSGSGSGTDFSLTISSLQAEDVAVYFCQQYYSI PNTFGQGTKLEIKR;

-   -   TRL1245 heavy chain variable region has the amino acid sequence:

(SEQ ID NO: 19) QVQLVESGGGLVKAGGSLRLSCVASGFTFSDYYMSWIRQAPGKGLEWISFISSSGDTIFYADSVKGRFTVSRDSAKNSLYLQMNSLKVEDTAVYYCARKG VSDEELLRFWGQGTLVTVSS;

-   -   TRL1245 light chain variable region has the amino acid sequence:

(SEQ ID NO: 20) DIVLTQDPSVSVSPGQTARITCSGDALPKKYAYWYQQKSGQAPVLVIYEDTKRPSGIPERFSGSSSGTVATLTISGAQVEDEADYYCYSTDSSGNQRVFG GGTKLTVL;

-   -   TRL1330 heavy chain variable region has the amino acid sequence:

(SEQ ID NO: 21) QVQLVESGTEVKNPGASVKVSCTASGYKFDEYGVSWVRQSPGQGLEWMGWISVYNGKTNYSQNFQGRLTLTTETSTDTAYMELTSLRPDDTAVYYCATDK NWFDPWGPGTLVTVSS;

-   -   TRL1330 light chain variable region has the amino acid sequence:

(SEQ ID NO: 22) DIVLTQSPSASGSPGQSITISCTGTNTDYNYVSWYQHHPGKAPKVIIYDVKKRPSGVPSRFSGSRSGNTATLTVSGLQTEDEADYYCVSYADNNHYVFGS GTKVTVL;

-   -   TRL1335 heavy chain variable region has the amino acid sequence:

(SEQ ID NO: 23) QVQLVESGAEVKKPGESLKISCKGSGYNFTSYWIGWVRQMPGKGLEWMGVIYPDDSDTRYSPSFKGQVTISADKSISTAFLQWSSLKASDTAVYHCARPP DSWGQGTLVTVSS;

-   -   TRL1335 light chain variable region has the amino acid sequence:

(SEQ ID NO: 24) DIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGLAPRLLIVGASNRATGIPARFSGSGSGTEFTLTISSLQSEDFAFYYCQQYNNWPFTFGP GTKVDVKR;

-   -   TRL1337 heavy chain variable region has the amino acid sequence:

(SEQ ID NO: 25) QVQLLESGPGLVKPSETPSLTCTVSGGSIRSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDMSKNQFSLKLSSVTAADTAMYYCARVYG GSGSYDFDYWGQGTLVTVSS;

-   -   TRL1337 light chain variable region has the amino acid sequence:

(SEQ ID NO: 26) DIVLTQSPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQLPGKAPKLMIYEVTKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCSSFAGSNNHV VFGGGTKLTVL;

-   -   TRL1338 heavy chain variable region has the amino acid sequence:

(SEQ ID NO: 27) QVQLTLRESGPTLVKPTQTLTLTCTFSGFSLSTNGVGVGWIRQPPGKALEWLAIIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTLTNMDPVDTGTYYCAHILGASNYWTGYLRYYFDYWGQGTLVTVST;

-   -   TRL1338 light chain variable region has the amino acid sequence:

(SEQ ID NO: 28) DIEMTQSPSVSVSPGQTARITCSGEPLAKQYAYWYQQKSGQAPVVVIYKDTERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYHCESGDSSGTYPVFG GGTKLTVL;

-   -   TRL1341 heavy chain variable region has the amino acid sequence:

(SEQ ID NO: 29) QVQLQESGGGLVQPGGSLKLSCAASGFIFSGSTMHWVRQASGKGLEWVGRIRSKTNNYATAYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCIS LPGGYSSGQGTLVTVSS;

-   -   TRL1341 light chain variable region has the amino acid sequence:

(SEQ ID NO: 30) DIMLTQPPSVSVSPGQTARITCSGDALPKKYTYWYQQKSGQAPVLVIYEDSKRPSEIPERFSAFTSWTTATLTITGAQVGDEADYYCYSTDITGDIGVFG GGTKLTVL;

-   -   TRL1347 heavy chain variable region has the amino acid sequence:

(SEQ ID NO: 31) QVQLVQSGGGLVQPGGSLKVSCVGSGFTFSASTIHWVRQASGKGLEWVGRIRSKANNYATVSAASLKGRFTISRDDSKNTAYLQVNSLKIEDTAIYYCTRPTACGDRVCWHGAWGQGTQVTVSP;

-   -   TRL1347 light chain variable region has the amino acid sequence:

(SEQ ID NO: 32) DIVLTQSPSASGTPGQRVTISCSGSRSNLGNNNVNWYQQLPGTAPKLLIFDNNERPSGVPGRFSGSKSGTSASLAISGLRSEDEADYYCASWDDSLNGWV FGGGTKVTVL;

-   -    and    -   TRL1361 heavy chain variable region has the amino acid sequence:

(SEQ ID NO: 33) QVQLVESGGGLAQPGGSLRLSCAASGFIFNTYAMGWVRQAPGKGLEWVSTVSAPGAGTYYTDSVKGRFIISRDNSKNILYLQMNRLRVEDTAVYYCARDQGGPAVAGARIFDYWGQGALVTVSS;

-   -   TRL1361 light chain variable region has the amino acid sequence:

(SEQ ID NO: 34) DIVLTQSPLSLSVTPGQPASISCKSSQSLLRSDGKTYLCWYLQKPGQPPQLLIYEVSNRVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSIQLR TFGQGTKVEIKR.

The encoding nucleotide sequences for the variable regions of theantibodies of the invention and are set forth in the sequence listing asfollows:

-   -   TRL1068: Heavy Chain: (SEQ ID NO:35); Light Chain: (SEQ ID        NO:36)    -   TRL1070: Heavy Chain: (SEQ ID NO:37); Light Chain: (SEQ ID        NO:38)    -   TRL1087: Heavy Chain: (SEQ ID NO:39); Light Chain: (SEQ ID        NO:40)    -   TRL1215: Heavy Chain: (SEQ ID NO:41); Light Chain: (SEQ ID        NO:42)    -   TRL1216: Heavy Chain: (SEQ ID NO:43); Light Chain: (SEQ ID        NO:44)    -   TRL1218: Heavy Chain: (SEQ ID NO:45); Light Chain: (SEQ ID        NO:46)    -   TRL1230: Heavy Chain: (SEQ ID NO:47); Light Chain: (SEQ ID        NO:48)    -   TRL1232: Heavy Chain: (SEQ ID NO:49); Light Chain: (SEQ ID        NO:50)    -   TRL1242: Heavy Chain: (SEQ ID NO:51); Light Chain: (SEQ ID        NO:52)    -   TRL1245: Heavy Chain: (SEQ ID NO:53); Light Chain: (SEQ ID        NO:54)    -   TRL1330: Heavy Chain: (SEQ ID NO:55); and codon optimized (SEQ        ID NO:56); Light Chain: (SEQ ID NO:57); and codon optimized (SEQ        ID NO:58)    -   TRL1335: Heavy Chain: (SEQ ID NO:59); Light Chain: (SEQ ID        NO:60)    -   TRL1337: Heavy Chain: (SEQ ID NO:61); Light Chain: (SEQ ID        NO:62)    -   TRL1338: Heavy Chain: (SEQ ID NO:63); Light Chain: (SEQ ID        NO:64)    -   TRL1341: Heavy Chain: (SEQ ID NO:65); Light Chain: (SEQ ID        NO:66)    -   TRL1347: Heavy Chain: (SEQ ID NO:67); Light Chain: (SEQ ID        NO:68)    -   TRL1361: Heavy Chain: (SEQ ID NO:69); Light Chain: (SEQ ID        NO:70).

EXAMPLE 2 Epitope Mapping

A set of 26 overlapping 15-mer peptides (offset by 3 residues) from theHU-beta of Staphylococcus aureus was synthesized, each with a biotin atthe N-terminus (followed by a short linker comprising SGSG). Peptideswere dissolved in DMSO (15-20 mg/mL), diluted 1:1000 in PBS and bound tostreptavidin coated plates in duplicate, to detect binding by the mAbs.These results are shown in FIG. 1.

TRL1068 and TRL1337 bound to peptides 19, SGSG AARKGRNPQTGKEID (SEQ IDNO:71) and 20, SGSG KGRNPQTGKEIDIPA (SEQ ID NO:72) strongly, and weaklyto peptide 18, SGSG RERAARKGRNPQTGK (SEQ ID NO:73). TRL1330 boundstrongly only to peptide 19. The epitope is thereby identified as withinKGRNPQTGKEIDI (SEQ ID NO:74).

TRL1338 binds strongly only to peptide 26, SGSGVPAFKAGKALKDAVK (SEQ IDNO:75). TRL1361 binds at a very different site, with strong binding topeptides 12 and 13 in the set, i.e., SGSG SLAKGEKVQLIGFGN (SEQ ID NO:76)and SGSG KGEKVQLIGFGNFEV (SEQ ID NO:77), and less strongly to peptide14, SGSG KVQLIGFGNFEVRER (SEQ ID NO:78). TRL1347 bound moderately topeptide 6, SGSG TKKEAGSAVDAVFES (SEQ ID NO:79).

TRL1335 binds to none of the 26 peptides; however, TRL1335 binds to theDNABII proteins from Pa and Sa. It is evident from these results thatTRL1335 binds to a conformational epitope. TRL1338 also binds stronglyto Pa and Sa. TRL1337 binds very strongly to the DNABII protein from Sa.

The epitope represented by SEQ ID NO:71 and SEQ ID NO:72 was then mappedmore precisely using 12-mer peptides offset by one residue across theregion defined by peptides 19 and 20 of the 3-residue offset set.Further, alanine scanning was conducted across this region, substitutingalanine for the native amino acid at each residue in turn. As shown inFIG. 3, the epitope for both TRL1068 AARKGRNPQTGEKEIDIPA (SEQ ID NO:80)and TRL1330 AARKGRNPQTGEKEIDIPA (SEQ ID NO:80) comprises as particularlyimportant the residues (underlined) in the linear sequence, which form aconformational epitope that is a beta-hairpin (anti-parallel beta sheet)structure. This structure has been extensively studied andpeptidomimetics for such structures have been prepared. Suchbeta-hairpin structures can be synthesized chemically in high yields andcan be cyclized to stabilize the conformation and to improve proteolyticstability. The definition of the epitopes for TRL1068 and TRL1330 athigh resolution thereby enables design of peptidomimetics suitable foruse as immunogens and as competing binding agents. These mAbs furtherprovide a useful way to measure likely utility of variants of the betahairpin structure. Only those variants that retain high affinity bindingto at least one of these reference mAbs are likely to be able to inducean immune response that provides the biofilm interfering activity of thereference mAbs.

As illustrated in FIG. 2, the region of the IHF or HU protein thatcorresponds to SEQ ID NO:80 is substantially conserved across multipleclinically important bacterial species. Structural modeling of IHF or HUfrom multiple species has confirmed that the homology is high,particularly in the DNA binding region (Swinger, K. K., et al., CurrentOpinion in Structural Biology (2004) 14:28-35). Peptides that onlypartially overlap with this optimal region are less likely to foldspontaneously into the relevant three dimensional conformation and willbe more difficult to chemically crosslink in order to lock in thatconformation. Optimizing the fidelity to the native protein in thismanner is advantageous for both mAb discovery and for use of the peptideas an immunogen.

Computational construction of IHF from E. coli shows that this epitopeis partially masked by DNA when bound. However, if exposed, theseportions of the proteins may generate antibodies of high affinitycapable of binding them and thus preventing the formation of biofilm orcausing an established biofilm to lose structural integrity as theDNABII protein is sequestered by the antibody. Other sites on the DNABIIprotein not involved in binding DNA may also suffice to achieveextraction of the protein out of the biofilm based on higher affinitybinding by the mAb as compared to the protein's affinity for componentsof the biofilm.

EXAMPLE 3 Determination of Affinity

For practice of the assay method, ˜1 mg of DNABII protein was required.DNABII protein is difficult to express in bacteria (since it has a dualfunction involving gene regulation, apparently leading to toxicity tobacteria when expressed at high levels). Obtaining sufficient materialfor mAb discovery from bacterial sources is thus difficult. The proteinwas therefore expressed in HEK293 (mammalian) cells, with apoly-histidine tag to enable easy purification. The homologs fromStaphylococcus aureus (Sa), Pseudomonas aeruginosa (Pa), Klebsiellapneumoniae (Kp), Acinetobacter baumannii (Ab) and Haemophilus influenzae(Hi) were all prepared in this manner. These five are of particularutility since they span a substantial portion of the diversity insequences of the DNABII family.

Antibodies TRL1068, 1330, 1333, 1337 and 1338 among them bind to theseproteins. Four of these, Sa, Kp, Ab, and Pa are members of theclinically problematic ESKAPE set, which are Enterobacter aerogenes,Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,Pseudomonas aeruginosa and Escherichia coli.

The ELISA assays were conducted as follows:

-   -   Plates were coated with 1 ug/ml of antigen in PBS overnight at        4° C.    -   Washed 4 times in PBS/0.05% Tween® 20.    -   Blocked in 3% BSA/PBS and stored until ready to use.    -   Washed 4 times in PBS/0.05% Tween® 20.    -   Incubated for 1 hr with serial dilutions of anti-DNABII mAb in        blocking buffer.    -   Washed 4 times in PBS/0.05% Tween® 20.    -   Incubated for 1 hr in 1 ug/ml of HRP-conjugated goat anti human        IgG Fc in blocking buffer.    -   Washed 4 times in PBS/0.05% Tween® 20.    -   Developed in TMB peroxidase substrate and color stopped with        stop solution with affinity estimated as the half-maximal        binding concentration.

The fitted curves from the ELISA points give a mid-point value thatrepresents an estimate of the affinity constant. The results forantibodies TRL's 1335, 1337, 1338, 1341, 1347 and 1361 in binding toHU-beta of S. aureus are shown in FIG. 4 and are summarized as follows.

affinity to TRL HU Sa 1335 >1 μM 1337 6 nM 1338 42 nM 1341 >1 μm1347 >500 nM 1361 6.6 nM

The results for TRL1068 and 1330 are shown in FIGS. 5A-5B and are asfollows:

TRL1068 TRL1330 Antigen Affinity (pM) Affinity (pM) P. aeruginosa 11 13S. aureus 15 23 K. pneumoniae 11 14 H. influenzae 5,000 26 Acinetobacterbaumannii 10 17

Although TRL1337 bound the same peptides used for epitope mapping inExample 2 as did TRL1068 and TRL1330, among the five full-length DNABIIproteins tested, TRL1337 bound only that of S. aureus. This result isfurther evidence for some conformational character to the epitopes.

EXAMPLE 4 In Vitro Bioactivity Assessment

TRL1068 was tested for bioactivity using a commercial assay fromInnovotech (Edmonton, Alberta; Canada). Biofilms were formed in multiplereplicates on plastic pegs in a 96-well microplate format exposed tomedia including Pseudomonas aeruginosa (ATCC 27853) or Staphylococcusaureus (ATCC 29213). Following biofilm formation, the pegs were treatedin different wells with saline or an isotype matched non-bindingantibody control or with TRL1068 at 1.2 μg/mL (˜10 nM) for 12 hours. Asevident in the scanning electron micrographs of the treated surfaces inFIGS. 6A and 6B, TRL1068 was highly effective at dissolving the biofilmas compared to the growth controls. These results establish that the mAbcan degrade the biofilm, thereby removing the attached bacteria. Itfurther shows that TRL1068 is active against biofilms produced by bothgram-positive and gram-negative bacteria. Such broad spectrum activityis rarely achieved by conventional antibiotics.

TRL1330 and TRL1361 were subjected to similar assays and providedsimilar results. TRL1330 was active against both Pseudomonas aeruginosaand Staphylococcus aureus and TRL1361 only against Staphylococcusaureus.

EXAMPLE 5 pH Dependence

The high affinity binding of TRL1068 was shown to be retained even asthe pH was decreased from physiological (pH 7.5) to pH 4.5, as shownbelow. FIGS. 7A-7C show the results for TRL1068 assessed against threedifferent DNABII homologs. In other studies, no significant change inbinding was seen at higher pH up to 8.5.

TRL1068 Affinity (nM) pH Sa Kp Pa 7.5 0.010 0.006 0.005 6.5 0.012 0.0060.005 5.5 0.012 0.009 0.006 4.5 0.012 0.050 0.009 3.5 0.36 3.0 0.26 2.53.5 2.6 2.1

Similarly, TRL1330 subjected to similar assays provides similar results.

This is important since the local micro-environment of infected tissuesis often at lower pH than in healthy tissues.

EXAMPLE 6 In Vivo Bioactivity Assessments

Several animal models exist for evaluation of activity. For example, asdescribed by investigators at University Hospital Basel (Switzerland), amodel for biofilm on implanted prostheses involves implanting Teflon®tissue cages (Angst+Pfister; Zurich, Switzerland) subcutaneously inBALB/c mice. Sterile perforated cylindrical Teflon® tissue cages,8.5×1×30 mm, 1.9 mL volume (Angst+Pfister AG, Zurich, Switzerland), wereimplanted subcutaneously into anesthetized mice. Upon complete woundhealing (two weeks), the mice were anesthetized and the cages tested forsterility by plating percutaneously aspirated tissue cage fluid (TCF) onColumbia sheep blood agar plates. To simulate a perioperative infection,700 colony-forming units (CFU) of MRSA (ATCC 43300) were injected intoeach cage (day 0). After 24 hours, the interior of the cage becomescoated with a biofilm. Treatment groups (given intraperitoneally (i.p.)starting 24 hours after the biofilm was formed): saline control,antibiotic alone (daptomycin (DAP), 50 mg/kg), or antibiotic incombination with TRL1068 (15 mg/kg). On days 2 and 3, the fluid withinthe tissue cage (TCF) was aspirated and plated to determine CFU ofplanktonic bacteria. At the end of the experiment, the animals weresacrificed and the implanted cage recovered. Following sonication torelease the adherent bacteria, the level of infection was again assessedas CFU per cage. As expected, the bacteria in the cage were onlypartially reduced by daptomycin alone. By contrast, TRL1068 incombination with daptomycin significantly reduced the bacteria insidethe cages on day 2 (P<0.01). Additionally, TRL1068 in combination withdaptomycin significantly reduced adherent bacteria inside the cage(P<0.02). See FIG. 8. Since daptomycin concentration in the bodydecreases substantially over 24 hours, a second experiment was conductedin which TRL1068 and daptomycin were administered by i.p. injectiondaily for 3 days. Similar efficacy at day 2 was observed, measuring theplanktonic bacteria in the cage fluid. At sacrifice, the remainingadherent bacteria level was again measured. A 2 log reduction wasobserved for the TRL1068 treated animals, an improvement over the 1 logreduction seen with only 1 dose of daptomycin. This result is consistentwith continuous erosion of the biofilm, releasing bacteria that can bekilled by daptomycin more effectively than when they are embedded in thebiofilm. Longer exposure to the antibody (over 5 days) was even moreeffective. Finally, including a pre-infection dose of TRL1068 in theregimen (including DAP+TRL1068 over 5 days) was highly effective ateliminating the biofilm. These results are described in: Estelles, A.,et al., Antimicrobial Agents Chemother. (2016) 60(4):2292-2301.

A second example is a model that involves inducing biofilm on heartvalves, mimicking native valve endocarditis (Tattevin, P., et al.,Antimicrob Agents Chemother (2013) 57:1157). New Zealand white rabbitsare anesthetized. The right carotid artery is cut and a polyethylenecatheter is positioned across the aortic valve and secured in place.Twenty four hours later, 1 mL of saline plus 8×10⁷ CFU of S. aureus isinjected through the catheter, which induces a biofilm infection in 95%of the animals. Drugs (anti-biofilm and antibiotic) are administeredi.v. and efficacy is evaluated after 4 days by tissue pathology andblood bacterial levels.

A third example is a rat model for valve infective endocarditis (IE).TRL1068 was evaluated in a catheter-induced aortic valve IE rat model(Xiong, Y. Q., et al., Antimicrob Agents Chemother. (2005) 49:3114-3121.Rats were infected with a clinical isolate (MRSA strain 300-169;associated with persistent bacteremia). Animals were treated with 6 daysof vancomycin (n=8; VAN: 120 mg/kg, SC, bid) alone or in combinationwith TRL1068 (n=10) or isotype control antibody (n=8); each at 15 mg/kg,i.v., QD on days 1 and 4. MRSA burden in cardiac vegetations,intracardiac catheter, kidney, spleen and liver was quantified atsacrifice on day 7 (mean log₁₀CFU/g tissue±SD). MRSA densities invegetations showed ≥1.75 log reduction in the TRL1068+VAN arm vs.isotype control+VAN arm (P<0.001). Significant reduction in CFU's wasalso observed for intracardiac catheters, kidneys, spleens and livers(P<0.05) for the VAN+TRL1068 arm vs. both VAN+isotype and isotype alonearms. A trend towards mortality reduction in the VAN+TRL1068 arm wasalso observed (P=0.09). Similarly, TRL1330 is subjected to these assaysto provide similar results.

Embodiments of the Invention

The various embodiments of the invention include a monoclonal bindingmoiety that has affinity for at least one DNABII protein that exceedsthe affinity of said DNABII protein for components of a biofilm thatincludes said DNABII protein, and includes embodiments wherein thebinding moiety is a monoclonal antibody (mAb), an aptamer, a non-Igscaffold or a structured short peptide, and those wherein said bindingmoiety binds an epitope on said DNABII protein that is conserved acrossbacterial species, and combinations of these features.

This embodiment includes those embodiments wherein binding moiety is anmAb and the mAb is an Fv antibody, a bispecific antibody, a chimericantibody, species-ized antibody or a complete antibody comprisinggeneric constant regions heterologous to variable regions, and thosewherein the biofilm component is branched DNA, and/or wherein the DNABIIprotein is IHF or a subunit thereof, or is HU protein or a subunitthereof, and those wherein said binding moiety dissolves biofilm derivedfrom at least two bacterial species including both Gram positive andGram negative species, including those wherein said species are S.aureus, P. aeruginosa, A. baumannii and K. pneumoniae, and those whereinthe binding moiety has affinity for biofilm-forming protein from atleast three bacterial species at least as strong as 100 pM, includingthose wherein said species are selected from S. aureus, P. aeruginosa,A. baumannii and K. pneumoniae, and wherein said affinity is at least asstrong as 40 pM, and wherein said species are S. aureus, P. aeruginosa,A. baumannii and K. pneumoniae.

Any of these embodiments may be mAbs which are humanized mAbs ormodified to be compatible with a feline, canine, equine, bovine,porcine, caprine or ovine species or wherein the variable and constantregions of said mAbs are human, feline, canine, equine, bovine, porcine,caprine or ovine.

Specific embodiments of the invention include binding moieties which aremAb or antigen binding fragments wherein the variable region comprises

-   -   (a) the CDR regions of the heavy chain of TRL1068 (SEQ ID NO:1);        or    -   (b) the CDR regions of the heavy chain of TRL1070 (SEQ ID NO:3);        or    -   (c) the CDR regions of the heavy chain of TRL1087 (SEQ ID NO:5);        or    -   (d) the CDR regions of the heavy chain of TRL1215 (SEQ ID NO:7);        or    -   (e) the CDR regions of the heavy chain of TRL1216 (SEQ ID NO:9);        or    -   (f) the CDR regions of the heavy chain of TRL1218 (SEQ ID        NO:11); or    -   (g) the CDR regions of the heavy chain of TRL1230 (SEQ ID        NO:13); or    -   (h) the CDR regions of the heavy chain of TRL1232 (SEQ ID        NO:15); or    -   (i) the CDR regions of the heavy chain of TRL1242 (SEQ ID        NO:17); or    -   (j) the CDR regions of the heavy chain of TRL1245 (SEQ ID        NO:19); or    -   (k) the CDR regions of the heavy chain of TRL1330 (SEQ ID        NO:21); or    -   (l) the CDR regions of the heavy chain of TRL1335 (SEQ ID        NO:23); or    -   (m) the CDR regions of the heavy chain of TRL1337 (SEQ ID        NO:25); or    -   (n) the CDR regions of the heavy chain of TRL1338 (SEQ ID        NO:27); or    -   (o) the CDR regions of the heavy chain of TRL1341 (SEQ ID        NO:29); or    -   (p) the CDR regions of the heavy chain of TRL1347 (SEQ ID        NO:31); or    -   (q) the CDR regions of the heavy chain of TRL1361 (SEQ ID        NO:33), or mAb or antigen binding fragments that compete for        binding DNABII protein with mAb or fragments comprising these        CDR regions.

With respect to the mAb in the previous paragraph, in some embodiments

-   -   the mAb of (a) further comprises the CDR regions of the light        chain of TRL1068 (SEQ ID NO:2); or    -   the mAb of (b) further comprises the CDR regions of the light        chain of TRL1070 (SEQ ID NO:4); or    -   the mAb of (c) further comprises the CDR regions of the light        chain of TRL1087 (SEQ ID NO:6); or    -   the mAb of (d) further comprises the CDR regions of the light        chain of TRL1215 (SEQ ID NO:8); or    -   the mAb of (e) further comprises the CDR regions of the light        chain of TRL1216 (SEQ ID NO:10); or    -   the mAb of (f) further comprises the CDR regions of the light        chain of TRL1218 (SEQ ID NO:12); or    -   the mAb of (g) further comprises the CDR regions of the light        chain of TRL1230 (SEQ ID NO:14); or    -   the mAb of (h) further comprises the CDR regions of the light        chain of TRL1232 (SEQ ID NO:16); or    -   the mAb of (i) further comprises the CDR regions of the light        chain of TRL1242 (SEQ ID NO:18); or    -   the mAb of (j) further comprises the CDR regions of the light        chain of TRL1245 (SEQ ID NO:20); or    -   the mAb of (k) further comprises the CDR regions of the light        chain of TRL1330 (SEQ ID NO:22); or    -   the mAb of (l) further comprises the CDR regions of the light        chain of TRL1335 (SEQ ID NO:24); or    -   the mAb of (m) further comprises the CDR regions of the light        chain of TRL1337 (SEQ ID NO:26); or    -   the mAb of (n) further comprises the CDR regions of the light        chain of TRL1338 (SEQ ID NO:28); or    -   the mAb of (o) further comprises the CDR regions of the light        chain of TRL1341 (SEQ ID NO:30); or    -   the mAb of (p) further comprises the CDR regions of the light        chain of TRL1347 (SEQ ID NO:32); or    -   the mAb of (q) further comprises the CDR regions of the light        chain of TRL1361 (SEQ ID NO:34), or mAb or antigen binding        fragments that compete for binding DNABII protein with mAb or        fragments comprising these CDR regions.

More specific embodiments are mAbs or, antigen binding fragments thereofwhich comprise

-   -   (a) the variable region of the heavy chain of TRL1068 (SEQ ID        NO:1); or    -   (b) the variable region of the heavy chain of TRL1070 (SEQ ID        NO:3); or    -   (c) the variable region of the heavy chain of TRL1087 (SEQ ID        NO:5); or    -   (d) the variable region of the heavy chain of TRL1215 (SEQ ID        NO:7); or    -   (e) the variable region of the heavy chain of TRL1216 (SEQ ID        NO:9); or    -   (f) the variable region of the heavy chain of TRL1218 (SEQ ID        NO:11); or    -   (g) the variable region of the heavy chain of TRL1230 (SEQ ID        NO:13); or    -   (h) the variable region of the heavy chain of TRL1232 (SEQ ID        NO:15); or    -   (i) the variable region of the heavy chain of TRL1242 (SEQ ID        NO:17); or    -   (j) the variable region of the heavy chain of TRL1245 (SEQ ID        NO:19); or    -   (k) the variable region of the heavy chain of TRL1330 (SEQ ID        NO:21); or    -   (l) the variable region of the heavy chain of TRL1335 (SEQ ID        NO:23); or    -   (m) the variable region of the heavy chain of TRL1337 (SEQ ID        NO:25); or    -   (n) the variable region of the heavy chain of TRL1338 (SEQ ID        NO:27); or    -   (o) the variable region of the heavy chain of TRL1341 (SEQ ID        NO:29); or    -   (p) the variable region of the heavy chain of TRL1347 (SEQ ID        NO:31); or    -   (q) the variable region of the heavy chain of TRL1361 (SEQ ID        NO:33), or mAb or antigen binding fragments that compete for        binding DNABII protein with mAb or fragments comprising these        variable regions; and in particular wherein    -   the mAb of (a) further comprises the variable region of the        light chain of TRL1068 (SEQ ID NO:2); or    -   the mAb of (b) further comprises the variable region of the        light chain of TRL1070 (SEQ ID NO:4); or    -   the mAb of (c) further comprises the variable region of the        light chain of TRL1087 (SEQ ID NO:6); or    -   the mAb of (d) further comprises the variable region of the        light chain of TRL1215 (SEQ ID NO:8); or    -   the mAb of (e) further comprises the variable region of the        light chain of TRL1216 (SEQ ID NO:10); or    -   the mAb of (f) further comprises the variable region of the        light chain of TRL1218 (SEQ ID NO:12); or    -   the mAb of (g) further comprises the variable region of the        light chain of TRL1230 (SEQ ID NO:14); or    -   the mAb of (h) further comprises the variable region of the        light chain of TRL1232 (SEQ ID NO:16); or    -   the mAb of (i) further comprises the variable region of the        light chain of TRL1242 (SEQ ID NO:18); or    -   the mAb of (j) further comprises the variable region of the        light chain of TRL1245 (SEQ ID NO:20); or    -   the mAb of (k) further comprises the variable region of the        light chain of TRL1330 (SEQ ID NO:22); or    -   the mAb of (l) further comprises the variable region of the        light chain of TRL1335 (SEQ ID NO:24); or    -   the mAb of (m) further comprises the variable region of the        light chain of TRL1337 (SEQ ID NO:26); or    -   the mAb of (n) further comprises the variable region of the        light chain of TRL1338 (SEQ ID NO:28); or    -   the mAb of (o) further comprises the variable region of the        light chain of TRL1341 (SEQ ID NO:30); or    -   the mAb of (p) further comprises the variable region of the        light chain of TRL1347 (SEQ ID NO:32); or    -   the mAb of (q) further comprises the variable region of the        light chain of TRL1361 (SEQ ID NO:34), or mAb or antigen binding        fragments that compete for binding DNABII protein with mAb or        fragments comprising these variable regions.

Each binding moiety, including the mAbs of the invention, binds to anepitope that is conserved with respect to a DNABII protein derived fromat least two or three or more species of bacteria. The epitope may belinear or conformational, and conservation of the epitope is determinedsimply by the ability of the binding moiety to successfully bind therelevant portion of the DNABII protein. Even for conformationalepitopes, the portion may constitute a limited region of the protein,and it is thus possible to identify conservation of the epitope byreview of the sequences of these proteins and selecting regions ofhomology.

The invention also includes pharmaceutical or veterinary compositionsfor treatment in a subject of a condition in said subject characterizedby formation of biofilms which comprises as active ingredient themonoclonal binding moiety as set forth in any of the foregoingembodiments in an amount effective to prevent or inhibit or dissolve abiofilm characteristic of said condition, said composition furtherincluding a suitable pharmaceutical excipient, including thosepharmaceutical or veterinary compositions which further include at leastone antibiotic, and/or further include at least one additional activeingredient.

The invention also includes a method to treat a condition in a subjectcharacterized by the formation of a biofilm in said subject or to detectthe formation of a biofilm in said subject, which method comprisestreating said subject with a binding moiety which is a monoclonalantibody (mAb), an aptamer, a non-Ig scaffold or a structured shortpeptide, or

-   -   wherein said binding moiety has affinity for at least one DNABII        protein that exceeds the affinity of said DNABII protein for        components of a biofilm that includes said DNABII protein;    -   wherein said binding moiety binds an epitope on said DNABII        protein that is conserved across bacterial species; and    -   wherein when the biofilm is to be detected, the method further        comprises observing complexation of said binding moiety with any        biofilm present, such as by employing a labeled binding moiety        such as a radioisotope, a dye, a fluorescent moiety or an enzyme        substrate.

Such conditions may be heart valve endocarditis, chronic non-healingwounds, including venous ulcers and diabetic foot ulcers, earinfections, sinus infections, urinary tract infections, pulmonaryinfections, cystic fibrosis, chronic obstructive pulmonary disease,catheter-associated infections, infections associated with implantedprostheses, periodontal disease, and Lyme disease.

In particular embodiments of these methods, the subject is human and thebinding moiety is an mAb which is a human or humanized mAb; inparticular wherein said binding moiety dissolves biofilm derived from atleast three bacterial species. Such species may be selected from S.aureus, P. aeruginosa, A. baumannii and K. pneumoniae.

In some embodiments of these methods, the binding moiety has affinityfor biofilm-forming protein from at least three bacterial species atleast as strong as 100 pM, wherein, in some embodiments, said speciesare S. aureus, P. aeruginosa, A. baumannii and K. pneumoniae.

In other embodiments, the binding moiety has affinity forbiofilm-forming protein from at least three bacterial species at leastas strong as 40 pM, in particular wherein said species are S. aureus, P.aeruginosa, A. baumannii and K. pneumoniae.

Particularly useful in the methods of the invention are those whereinthe binding moiety is an mAb or an antigen binding fragment thereof andwherein the variable region of said mAb comprises

-   -   (a) the CDR regions of the heavy chain of TRL1068 (SEQ ID NO:1);        or    -   (b) the CDR regions of the heavy chain of TRL1070 (SEQ ID NO:3);        or    -   (c) the CDR regions of the heavy chain of TRL1087 (SEQ ID NO:5);        or    -   (d) the CDR regions of the heavy chain of TRL1215 (SEQ ID NO:7);        or    -   (e) the CDR regions of the heavy chain of TRL1216 (SEQ ID NO:9);        or    -   (f) the CDR regions of the heavy chain of TRL1218 (SEQ ID        NO:11); or    -   (g) the CDR regions of the heavy chain of TRL1230 (SEQ ID        NO:13); or    -   (h) the CDR regions of the heavy chain of TRL1232 (SEQ ID        NO:15); or    -   (i) the CDR regions of the heavy chain of TRL1242 (SEQ ID        NO:17); or    -   (j) the CDR regions of the heavy chain of TRL1245 (SEQ ID        NO:19); or    -   (k) the CDR regions of the heavy chain of TRL1330 (SEQ ID        NO:21); or    -   (l) the CDR regions of the heavy chain of TRL1335 (SEQ ID        NO:23); or    -   (m) the CDR regions of the heavy chain of TRL1337 (SEQ ID        NO:25); or    -   (n) the CDR regions of the heavy chain of TRL1338 (SEQ ID        NO:27); or    -   (o) the CDR regions of the heavy chain of TRL1341 (SEQ ID        NO:29); or    -   (p) the CDR regions of the heavy chain of TRL1347 (SEQ ID        NO:31); or    -   (q) the CDR regions of the heavy chain of TRL1361 (SEQ ID        NO:33), or mAb or antigen binding fragments that compete for        binding DNABII protein with mAb or fragments comprising these        CDR regions; and including said mAb or fragment and in        particular wherein    -   the mAb of (a) further comprises the CDR regions of the light        chain of TRL1068 (SEQ ID NO:2); or    -   the mAb of (b) further comprises the CDR regions of the light        chain of TRL1070 (SEQ ID NO:4); or    -   the mAb of (c) further comprises the CDR regions of the light        chain of TRL1087 (SEQ ID NO:6); or    -   the mAb of (d) further comprises the CDR regions of the light        chain of TRL1215 (SEQ ID NO:8); or    -   the mAb of (e) further comprises the CDR regions of the light        chain of TRL1216 (SEQ ID NO:10); or    -   the mAb of (f) further comprises the CDR regions of the light        chain of TRL1218 (SEQ ID NO:12); or    -   the mAb of (g) further comprises the CDR regions of the light        chain of TRL1230 (SEQ ID NO:14); or    -   the mAb of (h) further comprises the CDR regions of the light        chain of TRL1232 (SEQ ID NO:16); or    -   the mAb of (i) further comprises the CDR regions of the light        chain of TRL1242 (SEQ ID NO:18); or    -   the mAb of (j) further comprises the CDR regions of the light        chain of TRL1245 (SEQ ID NO:20); or    -   the mAb of (k) further comprises the CDR regions of the light        chain of TRL1330 (SEQ ID NO:22); or    -   the mAb of (l) further comprises the CDR regions of the light        chain of TRL1335 (SEQ ID NO:24); or    -   the mAb of (m) further comprises the CDR regions of the light        chain of TRL1337 (SEQ ID NO:26); or    -   the mAb of (n) further comprises the CDR regions of the light        chain of TRL1338 (SEQ ID NO:28); or    -   the mAb of (o) further comprises the CDR regions of the light        chain of TRL1341 (SEQ ID NO:30); or    -   the mAb of (p) further comprises the CDR regions of the light        chain of TRL1347 (SEQ ID NO:32); or    -   the mAb of (q) further comprises the CDR regions of the light        chain of TRL1361 (SEQ ID NO:34), or mAb or antigen binding        fragments that compete for binding DNABII protein with mAb or        fragments comprising these CDR regions.

In more specific embodiments of the invention methods, the subject ishuman and said mAb or antigen-binding fragment comprises

-   -   (a) the variable region of the heavy chain of TRL1068 (SEQ ID        NO:1); or    -   (b) the variable region of the heavy chain of TRL1070 (SEQ ID        NO:3); or    -   (c) the variable region of the heavy chain of TRL1087 (SEQ ID        NO:5); or    -   (d) the variable region of the heavy chain of TRL1215 (SEQ ID        NO:7); or    -   (e) the variable region of the heavy chain of TRL1216 (SEQ ID        NO:9); or    -   (f) the variable region of the heavy chain of TRL1218 (SEQ ID        NO:11); or    -   (g) the variable region of the heavy chain of TRL1230 (SEQ ID        NO:13); or    -   (h) the variable region of the heavy chain of TRL1232 (SEQ ID        NO:15); or    -   (i) the variable region of the heavy chain of TRL1242 (SEQ ID        NO:17); or    -   (j) the variable region of the heavy chain of TRL1245 (SEQ ID        NO:19); or    -   (k) the variable region of the heavy chain of TRL1330 (SEQ ID        NO:21); or    -   (l) the variable region of the heavy chain of TRL1335 (SEQ ID        NO:23); or    -   (m) the variable region of the heavy chain of TRL1337 (SEQ ID        NO:25); or    -   (n) the variable region of the heavy chain of TRL1338 (SEQ ID        NO:27); or    -   (o) the variable region of the heavy chain of TRL1341 (SEQ ID        NO:29); or    -   (p) the variable region of the heavy chain of TRL1347 (SEQ ID        NO:31); or    -   (q) the variable region of the heavy chain of TRL1361 (SEQ ID        NO:33), or mAb or antigen binding fragments that compete for        binding DNABII protein with mAb or fragments comprising these        variable regions; and in particular wherein    -   the mAb of (a) further comprises the variable region of the        light chain of TRL1068 (SEQ ID NO:2); or    -   the mAb of (b) further comprises the variable region of the        light chain of TRL1070 (SEQ ID NO:4); or    -   the mAb of (c) further comprises the variable region of the        light chain of TRL1087 (SEQ ID NO:6); or    -   the mAb of (d) further comprises the variable region of the        light chain of TRL1215 (SEQ ID NO:8); or    -   the mAb of (e) further comprises the variable region of the        light chain of TRL1216 (SEQ ID NO:10); or    -   the mAb of (f) further comprises the variable region of the        light chain of TRL1218 (SEQ ID NO:12); or    -   the mAb of (g) further comprises the variable region of the        light chain of TRL1230 (SEQ ID NO:14); or    -   the mAb of (h) further comprises the variable region of the        light chain of TRL1232 (SEQ ID NO:16); or    -   the mAb of (i) further comprises the variable region of the        light chain of TRL1242 (SEQ ID NO:18); or    -   the mAb of (j) further comprises the variable region of the        light chain of TRL1245 (SEQ ID NO:20); or    -   the mAb of (k) further comprises the variable region of the        light chain of TRL1330 (SEQ ID NO:22); or    -   the mAb of (l) further comprises the variable region of the        light chain of TRL1335 (SEQ ID NO:24); or    -   the mAb of (m) further comprises the variable region of the        light chain of TRL1337 (SEQ ID NO:26); or    -   the mAb of (m) further comprises the variable region of the        light chain of TRL1338 (SEQ ID NO:28); or    -   the mAb of (o) further comprises the variable region of the        light chain of TRL1341 (SEQ ID NO:30); or    -   the mAb of (p) further comprises the variable region of the        light chain of TRL1347 (SEQ ID NO:32); or    -   the mAb of (q) further comprises the variable region of the        light chain of TRL1361 (SEQ ID NO:34), or mAb or antigen binding        fragments that compete for binding DNABII protein with mAb or        fragments comprising these variable regions.

As shown in Example 2, mAbs have been prepared that bind to aconformational epitope comprised in SEQ ID NO:80.

In other embodiments, the invention includes recombinant expressionsystems for producing any of the binding moieties listed above in caseswherein said binding moiety is a protein, wherein said expression systemcomprises a nucleotide sequence encoding said protein operably linked toheterologous control sequences for expression, and the invention alsoincludes recombinant host cells that have been modified to contain theseexpression systems and methods to prepare any of the proteinaceousbinding moieties set forth above which method comprises culturing thesecells.

In other embodiments, the invention includes methods to preventformation of or to dissolve a biofilm associated with an industrial orother non-physiological process which method comprises treating asurface susceptible to or containing a biofilm with any of the bindingmoieties described above.

The invention further includes methods to prepare an aptamer nucleicacid or nucleic acid mimic which method comprises preparing a nucleicacid or peptide-nucleic acid consisting of 10-25 nucleotides thatspecifically binds a specific binding partner to any of the monoclonalbinding moieties set forth above; especially when the specific bindingpartner is an epitope of a DNABII protein, and/or said epitope isconserved across at least three bacterial species.

The invention also includes an aptamer nucleic acid or peptide nucleicacid mimic prepared by the foregoing method.

Pharmaceutical or veterinary compositions for treatment in a subject ofa condition in said subject characterized by formation of biofilms whichcomprises as active ingredient the above aptamer in an amount effectiveto prevent or inhibit or dissolve a biofilm characteristic of saidcondition said composition further including a suitable pharmaceuticalexcipient, are also included.

The invention also includes a surface in an industrial or othernon-biological setting coated with any of the binding moieties describedabove or with the above aptamer described.

In another aspect, the invention includes a synthetic compound thatmimics the epitope to which TRL1068, TRL1330 or TRL1337, especiallyTRL1068 or TRL1330 binds, wherein the synthetic compound mimics theconformational epitope contained in SEQ ID NO:80. In some embodiments,the epitope comprises the sequence RNPQT (positions 6-10 of SEQ IDNO:80) from the HU-beta of S. aureus to which TRL1068 binds or thatcomprises the sequence KGRNPQTGKEI (positions 6-14 of SEQ ID NO:80) fromIHF of S. aureus HU-beta to which TRL1330 binds. The invention furtherincludes a method to obtain antisera effective to dissolve biofilm whichmethod comprises immunizing a subject with this synthetic compound andrecovering antiserum from said subject, as well as the polyclonalantiserum or monoclonal antibodies derived therefrom obtained from thissubject.

The mimics themselves can be verified by their ability to bind to theantibodies of the invention, especially TRL1068, TRL1330, TRL1337 orTRL1361. The mimics then, in turn, may be used to identify bindingmoieties from libraries of nucleic acids or other molecules that arecandidate binding agents, resulting in aptamers or other peptide-basedor small molecule-based binding moieties.

The invention also includes a method to treat biofilm-related conditionsin a subject, which method comprises administering to said subject theantiserum or these monoclonal antibodies.

The invention is also directed to a method to image a biofilm whichmethod comprises treating the biofilm with a monoclonal antibody orantigen-binding fragment thereof that binds specifically to an epitopewithin positions 5-20 of SEQ ID NO:76 or positions 5-20 of SEQ ID NO:77or of SEQ ID NO:78 or to a peptidomimetic of any of these, said antibodyconjugated to an observable label, and obtaining an image based on saidlabel, and also includes a method to measure the level of a DNABIIprotein which method comprises subjecting a sample in which said DNABIIprotein is to be detected to a sandwich assay in which one antibody orantigen-binding fragment thereof comprising said sandwich binds anepitope within positions 5-20 of SEQ ID NO:76 or positions 5-20 of SEQID NO:77 or positions 5-20 of SEQ ID NO:78 and the other antibody orantigen-binding fragment thereof in said sandwich binds to an epitopewithin positions 5-20 of any of SEQ ID NOS:71-75 and 79-80 or to apeptidomimetic of any of these.

The invention includes, specifically an mAb, which mAb is an Fvantibody, a bispecific antibody, a chimeric antibody, species-izedantibody or a complete antibody, wherein said complete antibodycomprises generic constant regions heterologous to variable regions,which mAb competes with TRL1335, TRL1338, TRL1341, TRL1347 or TRL1361for binding to a DNABII protein, in particular wherein the variableregion comprises

-   -   (a) the CDR regions of the heavy chain of TRL1335 (SEQ ID        NO:23); or    -   (b) the CDR regions of the heavy chain of TRL1338 (SEQ ID        NO:27); or    -   (c) the CDR regions of the heavy chain of TRL1341 (SEQ ID        NO:29); or    -   (d) the CDR regions of the heavy chain of TRL1347 (SEQ ID        NO:31); or    -   (e) the CDR regions of the heavy chain of TRL1361 (SEQ ID        NO:35), or more specifically    -   wherein    -   the mAb of (a) further comprises the CDR regions of the light        chain of TRL1335 (SEQ ID NO:24); or    -   the mAb of (b) further comprises the CDR regions of the light        chain of TRL1338 (SEQ ID NO:28); or    -   the mAb of (c) further comprises the CDR regions of the light        chain of TRL1341 (SEQ ID NO:30); or    -   the mAb of (d) further comprises the CDR regions of the light        chain of TRL1347 (SEQ ID NO:32); or    -   the mAb of (e) further comprises the CDR regions of the light        chain of TRL1361 (SEQ ID NO:34).

The invention also includes recombinant host cells that have beenmodified to contain a recombinant expression system wherein saidexpression system comprises one or more nucleotide sequences encodingone of the mAb's set forth above operably linked to one or moreheterologous control sequences for expression, and in particular whereinsaid mAb comprises a variable region encoded by nucleic acid isolatedfrom B cells of a human not immunized with DNABII protein or with afragment thereof, as well as a method to prepare an mAb that binds aDNABII protein which method comprises culturing these cells.

The invention further includes a method to prevent formation of, or todissolve a biofilm associated with, a non-physiological process whichmethod comprises treating a surface associated with said processsusceptible to, or containing a, biofilm with an effective amount of theforegoing mAbs, and a non-physiological surface coated with one or moreof these mAbs.

The invention claimed is:
 1. A monoclonal antibody (mAb), which mAb isan Fv antibody, a bispecific antibody, a chimeric antibody, species-izedantibody or a complete antibody, wherein said complete antibodycomprises generic constant regions heterologous to the variable regionsthereof, wherein said mAb a) has affinity for at least one DNA bindingprotein II (DNABII) protein that exceeds the affinity of said DNABIIprotein for components of a biofilm that includes said DNABII proteinand binds DNABII protein from at least three bacterial species; and b)binds to a conformational epitope comprised in SEQ ID NO:80; and c)wherein the variable region comprises the heavy and light chain variableregion of TRL1068 (SEQ ID NO:1) and (SEQ ID NO: 2) or of TRL1330 (SEQ IDNO:21) and (SEQ ID NO: 22) or of TRL1337 (SEQ ID NO:25) and (SEQ IDNO:26).
 2. A pharmaceutical or veterinary composition which comprises asan active ingredient the mAb of claim 1 along with a suitablepharmaceutical excipient.
 3. The pharmaceutical or veterinarycomposition of claim 2 which further includes at least one antibiotic,and/or which further includes at least one additional active ingredientwhich is an immunostimulant and/or an antipyrogenic and/or an analgesic.4. A monoclonal antibody (mAb) of claim 1, which comprises the heavy andlight chain variable region of TRL1068 (SEQ ID NO:1) and (SEQ ID NO: 2).5. A pharmaceutical or veterinary composition which comprises as anactive ingredient the mAb of claim 4 along with a suitablepharmaceutical excipient.
 6. The pharmaceutical or veterinarycomposition of claim 5 which further includes at least one antibiotic,and/or which further includes at least one additional active ingredientwhich is an immunostimulant and/or an antipyrogenic and/ or ananalgesic.